Site specific homogeneous with KSP inhibitors

ABSTRACT

The invention relates to site specific homogeneous binder drug conjugates of kinesin spindle protein inhibitors, to active metabolites of these conjugates, to processes for preparing these conjugates, to the use of these conjugates for the treatment and/or prophylaxis of diseases and to the use of these conjugates for preparing medicaments for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders such as, for example, cancer diseases. Such treatments can be carried out as monotherapy or else in combination with other medicaments or further therapeutic measures.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of the U.S. National Stage application Ser. No. 15/739,130, filed Dec. 21, 2017, and claims priority to International Application No. PCT/EP2016/064120, filed internationally on Jun. 20, 2016, which claims the benefit of European Application Nos. 16160780.9, filed Mar. 16, 2016, and 15173488.6, filed Jun. 23, 2015, all of which are incorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 777052019300seqlist.txt, date recorded: Dec. 18, 2017, size: 405 KB).

INTRODUCTION AND STATE OF THE ART

The invention relates to site specific homogeneous binder drug conjugates of kinesin spindle protein inhibitors, to active metabolites of these conjugates, to processes for preparing these conjugates, to the use of these conjugates for the treatment and/or prophylaxis of diseases and to the use of these conjugates for preparing medicaments for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders such as, for example, cancer diseases. Such treatments can be carried out as monotherapy or else in combination with other medicaments or further therapeutic measures.

Cancer diseases are the consequence of uncontrolled cell growth of the most diverse tissues. In many cases, the new cells penetrate into existing tissue (invasive growth), or they metastase into remote organs. Cancer diseases occur in the most diverse organs and often have tissue-specific courses of the disease. The term cancer as a generic term therefore describes a large group of defined diseases of various organs, tissue and cell types.

Tumours in early stages can possibly be removed by surgical and radiotherapy measures. Metastased tumours as a rule can only be treated palliatively by chemotherapeutics. The aim here is to achieve the optimum combination of an improvement in the quality of life and prolonging of life.

Conjugates of binder proteins with one or more active compound molecules are known, in particular in the form of antibody drug conjugates (ADCs) in which an internalising antibody directed against a tumour-associated antigen is covalently attached via a linker to a cytotoxic agent. Following introduction of the ADCs into the tumour cell and subsequent dissociation of the conjugate, either the cytotoxic agent itself or a cytotoxic metabolite formed therefrom is released within the tumour cell and can unfold its action therein directly and selectively. In this manner, in contrast to conventional chemotherapy, damage to normal tissue is contained in significantly narrower limits [see, for example, J. M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); A. M. Wu and P. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005); P. D. Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B. Stump, Bioconjugate Chem. 21, 5-13 (2010)]. Thus, WO2012/171020 describes ADCs in which a plurality of toxophor molecules are attached via a polymeric linker to an antibody. As possible toxophors, WO2012/171020 mentions, among others, the substances SB 743921, SB 715992 (Ispinesib), MK-0371, AZD8477, AZ3146 and ARRY-520.

The substances mentioned last are kinesin spindle protein inhibitors. Kinesin spindle protein (KSP, also known as Eg5, HsEg5, KNSL1 or KIF11) is a kinesin-like motorprotein which is essential for the bipolar mitotic spindle to function. Inhibition of KSP leads to mitotic arrest and, over a relatively long term, to apoptosis (Tao et al., Cancer Cell 2005 Jul. 8(1), 39-59). After the discovery of the first cell-penetrating KSP inhibitor, Monastrol, KSP inhibitors have established themselves as a class of novel chemotherapeutics (Mayer et al., Science 286: 971-974, 1999), and they are subject of a number of patent applications (e.g. WO2006/044825; WO2006/002236; WO2005/051922; WO2006/060737; WO03/060064; WO03/040979; and WO03/049527). However, since KSP unfolds its action only during a relatively short period of time during the mitosis phase, KSP inhibitors have to be present in a sufficiently high concentration during these initial phases.

Antibody conjugation methods typically include chemical reaction of lysines or cysteines with activated esters or maleimide functionality respectively. However, these reactions are difficult to control with regard to site-specificity and stoechiometry, which leads to heterogeneous products (Wang et al., Protein Sci. 14, 2436-2446 (2005); Hamblett et al., Clin. Cancer Res. 10, 7063-7070 (2004); Sun et al., Bioconjug. Chem. 16, 1282-1290 (2005); Willner et al., Bioconjug. Chem. 4, 521-527 (1993)). Consequently, heterogeneous ADCs may contain both unconjugated and overloaded antibodies. Unconjugated antibodies compete with drug-loaded species for antigen binding that can diminish the activity of ADC therapeutics. On the other hand, a high degree of the antibody modification may result in antibody aggregation, increased toxicity, decreased stability and shorter half-life of ADCs in the circulation (Sochaj et al., Biotechnology Advances, 33, 775-784 (2015)). It has been reported that heterogeneity of ADC species can influence its pharmacokinetics (PK), in vivo performance and safety profiles (Jackson et al., PLoS One 9, e83865 (2014); Junutula et al., Nat. Biotechnol. 26, 925-932 (2008); Strop et al., Chem. Biol. 20, 161-167 (2013); Boswell et al., Bioconjugate Chem. 22, 1994-2004 (2011)).

In addition, batch to batch consistency in ADC production is challenging and requires diligent manufacturing capabilities. Therefore regulatory requirements may change in the future for the approval of new ADCs.

Site specific conjugation, in which a known number of linker-drugs are consistently conjugated to defined sites, is one way to overcome these challenges. Heterogeneity is minimized and ADC properties are more predictable, with consistent conjugate production from batch to batch. Drug-to-antibody ratio (DAR) is precisely controlled and can be tailored to various linker-drugs. There are various methods described in literature for site specific conjugation (Agarwal et al., Bioconjug. Chem. 26, 176-192 (2015); Cal et al., Angew. Chem. Int. Ed. Engl. 53, 10585-10587 (2014); Behrens et al., MAbs 6, 46-53 (2014); Panowski et al., MAbs 6, 34-45 (2014)). Methods for site specific conjugation include, in particular, enzymatic methods, e.g using transglutaminases (TGases), glycyltransferases or formylglycine generating enzyme (Sochaj et al., Biotechnology Advances, 33, 775-784 2015).

In WO2014/198817 anti-TWEAKR antibodies and in WO2015/189143 aglycosylated anti-TWEAKR antibodies are described that can used in antibody drug conjugates (ADCs).

Further, in WO2015/096982 antibody drug conjugates (ADCs) are described with kinesin spindel Protein (KSP). Especially this application describes ADCs with a TWEAKR antibody.

The present invention provides new site specific homogeneous binder conjugates of kinesin spindle protein inhibitors where the kinesin spindle protein inhibitors are conjugated to glutamine side chains of the binder and lacking the described disadvantages of randomly coupled binder drug conjugates.

More specifically the present invention provides new site specific homogeneous binder conjugates of kinesin spindle protein inhibitors where the kinesin spindle protein inhibitors are conjugated to glutamine side chains of the binder and lacking the described disadvantages of randomly coupled binder drug conjugates using transglutaminases (TGases).

SUMMARY OF THE INVENTION

Against this background it is an object of the present invention to provide substances which, after administration at a relatively low concentration, unfold apoptotic action and may therefore be of benefit for cancer therapy.

To achieve this object, the invention provides site specific homogenous conjugates of a binder or derivatives thereof with one or more active compound molecules, the active compound molecule being one or more kinesin spindle protein inhibitor (KSP inhibitor) or a prodrug thereof, attached to the binder via a linker L. The binder is preferably a binder protein or peptide, particularly preferably a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or an antigen-binding fragment thereof or an anti-EGFR antibody or an antigen-binding fragment thereof. Particular preference is given to an anti-TWEAKR antibody which binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), in particular the anti-TWEAKR antibody TPP-2090, or the anti-Her2 antibody trastuzumab. If the binder is an antibody it comprises an acceptor glutamine, preferentially in the constant region. Such acceptor glutamines can be introduced by mutations of suitable positions into glutamine (e.g. mutation N297Q, Kabat EU numbering) or by generation of deglycosylated or aglycosylated antibodies (e.g. by enzymatic deglycosylation by PNGase F or by mutation of N297X, Kabat EU numbering). In that later case of a deglycosylated or an aglycosylated antibody the glutamine Q295 (Kabat EU numbering) becomes an acceptor glutamine. Highly preferred is an antibody comprising a mutation N297A or N297Q (Kabat EU numbering). Therefore in general, antibodies described here also include aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering system of antibodies, see Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) of the heavy chain to any amino acid. Furthermore antibodies described here also include variants of the described antibodies being engineered to contain one or more acceptor glutamine residues for transglutaminase (TGase) catalyzed reactions.

One way for this attachment are literature-described approaches dealing with a site specific conjugation of binders using transglutaminase. Transglutaminases (TGase) including bacterial transglutaminase (BTG) (EC 2.3.2.13) are a family of enzymes that catalyze the formation of a covalent bond between the γ-carbonyl amide group of glutamines and the primary amine of lysines. Since some TGases also accept substrates other than lysine as the amine donor, they have been used to modify proteins including antibodies at suitable acceptor glutamine residues (Jeger et al., Angewandte Chemie Int. Ed. Engl 49, 9995-9997 (2010); Josten et al., J. Immunol. Methods 240, 47-54 (2000); Mindt et al., Bioconjugate Chem. 19, 271-278 (2008); Dennler et al., in Antibody Drug Conjuagtes (Ducry, L., Ed.), pp 205-215, Humana Press. (2013)). On the one hand transglutaminases were used for coupling of drugs to antibodies bearing genetically artificial glutamine tags being transglutaminse acceptor glutamines introduced by genetically engineering (Strop et al., Chem. Biol. 20, 161-167 (2013)). On the other hand it was reported that the conserved glutamine Q295 (Kabat numbering system of IgGs) located in the constant domain of the heavy chain is the sole γ-carbonyl amide donor for bacterial transglutaminase (EC 2.3.2.13) within the backbone of a aglycosylated IgG1, whereas no acceptor glutamine is present in the backbone in IgG1 being glycosylated at position N297 (kabat numbering) of the heavy chain (Jeger et al., Angewandte Chemie Int. Ed. Engl 49, 9995-9997 (2010)). In summary, the bacterial transglutaminase can be used for the conjugation of an amine group of the linker/drug to an acceptor glutamine residue of the antibody.

Such acceptor glutamines can be introduced by engineering of the antibody by mutations or by generation of aglycosylated antibodies. Such aglycosylated antibodies can be generated by deglycosylation using N-glycosidase F (PNGaseF) or by mutation of the N297 (Kabat numbering) of the glycosylation site of the heavy chain to any other amino acid. Enzymatic conjugation of such aglycosylated antibodies was described for aglycosylated antibody variants bearing the mutations N297D, N297Q (Jeger et al., Angewandte Chemie Int. Ed. Engl 49, 9995-9997 (2010)), or N297S (see patent applications WO2013092998A1 and WO2013092983A2). Enzymatic conjugation using transglutaminase of such aglycosylated antibodies provides ADCs with DAR of 2 in general, in which both heavy chains are functionalized site specifically at position Q295 (Kabat numbering). The mutation N297Q of the antibody provides 1 additional site for conjugation at each heavy chain leading for example to ADCS with DAR of 4, in which both heavy chains are functionalized site-specifically at position Q295 and Q297 (Kabat numbering). Antibody variants bearing the mutations Q295N and N297Q provide one acceptor glutamine residue at position Q297 (Simone Jeger, Site specific conjugation of tumour targeting antibodies using transglutaminase, Dissertation at ETH Zurich (2009)). There are several examples in literature describing site specific conjugation of aglycosylated antibodies via transglutaminase (e.g. Dennler et al., Bioconjugate Chemistry 19, 569-578 (2014); Lhospice et al., Molecular Pharmaceutics 12, 1863-1871 (2015)). The strategy using transglutaminase catalyzed conjugation of aglycosylated antibodies is summarized in FIG. 8 .

The inventors have found a way to attach the binder to the KSP inhibitor in a site specific homogenous manner in order to achieve the object mentioned above. Furthermore they demonstrated that transglutaminase could efficiently catalyse conjugation to aglycosylated antibody variants bearing the mutation N297A (Kabat numbering).

The invention provides site specific homogeneous conjugates of a binder or derivative thereof with one or more active compound molecules, the active compound molecule being a kinesin spindle protein inhibitor (KSP inhibitor) attached to the binder via a linker L and the linker L being conjugated to a specific site at the binder, preferably a glutamine side chain of the binder.

More specifically the invention provides site specific homogeneous conjugates of a binder or derivative thereof with one or more active compound molecules, the active compound molecule being a kinesin spindle protein inhibitor (KSP inhibitor) attached to the binder via a linker L and the linker L being conjugated to a glutamine side chain of the binder using transglutaminases (TGases).

The conjugate according to the invention can be represented by the general formula BINDER

L

KSP]_(m)]_(n) where BINDER represents the binder, preferably an antibody, L represents the linker, KSP represents the KSP inhibitor, m represents a number from 1 to 5, preferably 1, and n represents 2 to 10, preferably 2 to 4, and also preferably 2 or 4. Here, m is the number of KSP inhibitors per linker and n the number of KSP inhibitor/linker conjugates per BINDER. The sum of all KSP present in the conjugate is thus the product of m and n. The binder is preferably a binder peptide or protein such as, for example, an antibody. If the binder is an antibody it comprises an acceptor glutamine, preferentially in the constant region. Such acceptor glutamines can be introduced by mutations of suitable positions into glutamine (e.g. mutation N297Q, Kabat EU numbering) or by generation of deglycosylated or aglycosylated antibodies (e.g. by enzymatic deglycosylation by PNGase F or by mutation of N297X, Kabat EU numbering). In that later case of a deglycosylated or an aglycosylated antibody the glutamine Q295 (Kabat EU numbering) becomes an acceptor glutamine. Highly preferred is an antibody comprising a mutation N297A or N297Q (Kabat EU numbering). Furthermore, the linker is attached to glutamine residues of the binder peptide or protein or derivative thereof. Particular preference is given to binding to glutamine residues of an antibody.

According to the invention, the kinesin spindle protein inhibitors may have the substructure I(sub) below:

-   -   where     -   #a represents a bond to the remainder of the molecule;     -   R^(1a) represents —H, -MOD, or —(CH₂)₀₋₃Z,         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or             —CH(CH₂W)Z′,         -   where Y³ represents H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C═O—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NHC(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R^(2a) represents —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R^(4a) represents H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NHC(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R^(4a) represents a group of         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or the         cathepsin cleavable group or a group         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-,         -   where R²¹ represents C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group,             which may be substituted one or more times with —NH₂,             —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,             —N(alkyl)-C(═O)-alkyl, —SO₃H, —SO₂NH₂, —SO₂—N(alkyl)₂,             —COOH, —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or OH; or which             represents —H or a group —(O)_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1,         -   where v is a number from 1 to 10,         -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂);         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His or one of the             corresponding N-alkyl amino acids, preferably N-methyl amino             acids; or     -   R^(2a) and R^(4a) together represent (with formation of a         pyrrolidine ring) CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, halogen (preferably —F or —Cl),             —NH₂, —COOH, —SO₃H, —SH C₁₋₄-alkyl, Halo-C₁₋₄-alkyl,             C₁₋₄-alkoxy, hydroxy substituted C₁₋₄-alkyl,             —C(═O)—O—(C₁₋₄-alkyl) or —OH.

According to the invention, the kinesin spindle protein inhibitor may be attached to the binder via a linker by substitution of a hydrogen atom at R^(1a), R^(2a), R^(4a) or R¹⁰.

The KSP inhibitor which is attached to this binder (or the KSP inhibitors, since frequently more than one KSP inhibitor is attached to the binder), is preferably a compound of the formula (Ia) below:

-   -   where     -   R^(1a) represents —H, -MOD or —(CH₂)₀₋₃Z,         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or             —CH(CH₂W)Z′,         -   Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NHC(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R^(2a) represents —H, -MOD, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where -MOD is represented as defined infra,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R^(4a) represents —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂, and         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R^(4a) represents a group of         -   R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—, or         -   R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)—, or the             cathepsin cleavable group or formula             R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-,         -   where R²¹ represents C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group             (which may be substituted one or more times with —NH₂,             —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,             —N(alkyl)-C(═O)-alkyl, —SO₃H, —S(═O)₂NH₂, —S(═O)₂—N(alkyl)₂,             —COOH, —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or OH), —H or a group             —(O)_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1 and         -   where v is a number from 1 to 20,         -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂);         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His or one of the             corresponding N-alkyl amino acids, preferably N-methyl amino             acids;         -   or     -   R^(2a) and R^(4a) together represent (with formation of a         pyrrolidine ring) CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, halogen (preferably —F or —Cl),             —NH₂, —COOH, —SO₃H, —SH, C₁₋₄-alkyl halo-C₁₋₄-alkyl             C₁₋₄-alkoxy, hydroxy substituted C₁₋₄-alkyl,             —C(═O)—O—(C₁₋₄-alkyl) or —OH;     -   R^(3a) represents -MOD or an optionally substituted alkyl,         cycloalkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl         group, preferably a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or C₆₋₁₀-aralkyl,         C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where -MOD is represented as defined infra,         -   where n is 0, 1 or 2,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NHC(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH     -   R^(8a) represents C₁₋₁₀-alkyl or (CH₂)₀₋₂—(HZ²),         -   where HZ² represents a 4- to 7-membered heterocycle having             up to two heteroatoms selected from the group consisting of             N, O and S;     -   HZ represents a mono- or bicyclic heterocycle which may be         substituted by one or more substituents selected from the group         consisting of halogen, C₁₋₁₀-alkyl groups, C₆₋₁₀-aryl groups and         C₆₋₁₀-aralkyl groups which may optionally be substituted by         halogen;     -   -MOD as defined supra represents —(NR¹⁰)n-(G1)o-G2-G3,         -   where R¹⁰ represents —H; halogen or C₁-C₃-alkyl;         -   where G1 represents NHC(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents NHC(═O)— or

-   -   -    R¹⁰ does not represent NH₂);         -   n is 0 or 1;         -   o is 0 or 1;         -   G2 represents a linear and/or branched hydrocarbon group             which has 1 to 10 carbon atoms and which may be interrupted             once or more than once by one or more of the groups             -   —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,                 —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NRyNRy-,                 —C(═O)—NR^(y)NR^(y)—, —C(═O)—, —CR^(x)═N—O—,             -   and where the hydrocarbon group including any side                 chains may be substituted by —NHC(═O)—NH₂, —COOH, —OH,                 —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or                 sulphonic acid,         -   where R^(y) represents H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NHC(═O)—NH₂, —COOH, —OH, —NH₂, NH—CNNH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl),         -   where G3 represents —H or —COOH,         -   where -MOD preferably has at least one group —COOH;         -   and where the kinesin spindle protein inhibitor is attached             to the linker by substitution of a hydrogen atom at R^(1a),             R^(2a), R^(3a), R^(4a), R^(8a) or R¹⁰ or optionally via one             of the substituents of HZ, in particular via R^(1a), R^(2a),             R^(3a), R^(4a) or R¹⁰,         -   and the salts, solvates, salts of the solvates, and epimers             thereof.

    -   KSP-L- in the above shown formula has preferably the following         formula (IIa):

-   -   -   where         -   X₁ represents N, X₂ represents N and X₃ represents C; or         -   X₁ represents N, X₂ represents C and X₃ represents N; or         -   X₁ represents CH or CF, X₂ represents C and X₃ represents N;             or         -   X₁ represents NH, X₂ represents C and X₃ represents C; or         -   X₁ represents CH, X₂ represents N and X₃ represents C         -   (with X₁ representing CH, X₂ representing C and X₃             representing N being preferred);         -   R¹ represents H, -L-#1, MOD or —(CH₂)₀₋₃Z,             -   where -L-#1 represents the linker and #1 represents the                 bond to the binder or derivative thereof             -   where -MOD is represented as defined infra,             -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,                 —C(═O)—NY¹Y² or —C(═O)—OY³,             -   where Y¹ and Y² independently of one another represent                 —H, —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or                 —CH(CH₂W)Z′,             -   where Y³ represents —H or —(CH₂)₀₋₃Z′,             -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,                 —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or                 —(C(═O)—NH—CHY⁴)₁₋₃COOH,             -   where W represents —H or —OH,             -   where Y⁴ represents linear or branched C₁₋₆-alkyl which                 is optionally substituted by                 -   NHC(═O)—NH₂, or represents aryl or benzyl which are                     optionally substituted by —NH₂;         -   R² represents -L-#1, H, -MOD, —C(═O)—CHY⁴—NHY⁵ or             —(CH₂)₀₋₃Z,             -   where -L-#1 represents the linker and #1 represents the                 bond to the binder or derivative thereof,             -   where -MOD is represented as defined infra,             -   where Z represents —H, halogen, —OY³, —SY³, NHY³,                 —C(═O)—NY¹Y² or —C(═O)—OY³,             -   where Y¹ and Y² independently of one another represent                 —H, —NH₂ or —(CH₂)₀₋₃Z′, and             -   where Y³ represents —H or —(CH₂)₀₋₃Z′,             -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;             -   where Y⁴ represents linear or branched C₁₋₆-alkyl which                 is optionally substituted by —NHC(═O)—NH₂, or represents                 aryl or benzyl which are optionally substituted by —NH₂,             -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,             -   where Y⁶ represents linear or branched C₁₋₆-alkyl;         -   R⁴ represents -L-#1, —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,             -   where -L-#1 represents the linker and #1 represents the                 bond to the binder or derivative thereof,             -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y²                 or —C(═O)—OY³,             -   where Y¹ and Y² independently of one another represent                 —H, —NH₂ or —(CH₂)₀₋₃Z′,             -   where Y³ represents —H or —(CH₂)₀₋₃Z′,             -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;             -   where Y⁴ represents linear or branched C₁₋₆-alkyl which                 is optionally substituted by                 -   —NHC(═O)—NH₂, or represents aryl or benzyl which are                     optionally substituted by —NH₂, and             -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,             -   where Y⁶ represents linear or branched C₁₋₆-alkyl,         -   or         -   R⁴ represents a group of         -   R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or         -   R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)— or the             cathepsin cleavable group R²¹—(C═O)₍₀₋₁₎—(P3)₍₁₋₂₎-P2-,             -   where R²¹ represents H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or                 C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,                 C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,                 heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,                 C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,                 C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy                 group (which may be substituted one or more times with                 —NH₂, —NH-alkyl, —N(alkyl)₂,                 -   —NH—C(═O)-alkyl, N(alkyl)-C(═O)-alkyl, —SO₃H,                     —SO₂NH₂, —S(═O)₂—N(alkyl)₂, —COOH, —C(═O)—NH₂,                     —C(═O)—N(alkyl)₂, or OH), —H or a group                     —(O)_(x)—(CH₂CH₂O)_(y)—R²²,             -   where x is 0 or 1 and             -   where v is a number from 1 to 20,             -   where R²² represents —H, -alkyl (preferably                 C₁₋₁₂-alkyl), —CH₂—COOH, —CH₂—CH₂—COOH, or                 —CH₂—CH₂—NH₂);             -   where P2 is an amino acid selected from Gly, Pro, Ala,                 Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys,                 Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His;             -   where P3 is an amino acid independently selected from                 Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp,                 Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline                 and His or one of the corresponding N-alkyl amino acids,                 preferably N-methyl amino acids; in case there is more                 than one amino acid P3, P3 may have the same or                 different amino acids, as defined above;             -   or         -   R² and R⁴ together (with formation of a pyrrolidine ring)             represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,             -   where R¹⁰ represents L-#1, —H, —NH₂, —SO₃H, —COOH, —SH                 or —OH, and wherein the hydrogen atom of the secondary                 amino group in the pyrrolidine ring may be replaced by                 R²¹—C(═O)—P3₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—SIG-;             -   where L-#1 represents the linker and #1 represents the                 bond to the binder or derivative thereof,             -   where P2 is an amino acid selected from Gly, Pro, Ala,                 Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys,                 Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His;             -   where P3 is an amino acid independently selected from                 Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp,                 Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline                 and His or one of the corresponding N-alkyl amino acids,                 preferably N-methyl amino acids;             -   where -SIG is a self-immolative group, which, upon                 cleavage of the C(═O)—SIG bond provides the free                 secondary amine;         -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or             C(═N—NH₂)—;         -   R³ represents -L-#1, -MOD or an optionally substituted             alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl,             heterocycloalkyl group, preferably -L-#1 or a C₁₋₁₀-alkyl,             C₆₋₁₀-aryl or C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or C₅₋₁₀-heterocycloalkyl group             which may be substituted by 1-3 —OH groups, 1-3 halogen             atoms, 1-3 halogenated alkyl groups (each having 1-3 halogen             atoms), 1-3 —O-alkyl groups, 1-3 —SH groups, 1-3 —S-alkyl             groups, 1-3 —O—C(═O)-alkyl groups, 1-3 —O—C(═O)—NH-alkyl             groups, 1-3 —NH—C(═O)-alkyl groups, 1-3 —NH—C(═O)—NH-alkyl             groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3 —S(═O)₂—NH-alkyl             groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂ groups, 1-3             —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,             -   where -L-#1 represents the linker and #1 represents the                 bond to the binder or derivative thereof,             -   where -MOD is represented as defined infra,             -   where n represents 0, 1 or 2,             -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,                 —C(═O)—NY¹Y² or —C(═O)—OY³,             -   where Y¹ and Y² independently of one another represent                 —H, —NH₂ or —(CH₂)₀₋₃Z′ and             -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NHC(═O)—CH₃)Z′,                 —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′, where             -   where Z′ represents —H, —SO₃H, —NH₂ or COOH         -   R⁵ represents -L-#1, —H, —NH₂, —NO₂, halogen (in particular             —F, —Cl, —Br), —CN, —CF₃, —OCF₃, —CH₂F, —CH₂F, —SH or             —(CH₂)₀₋₃Z,             -   where -L-#1, represents the linker and #1 represents the                 bond to the binder or derivative thereof,             -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,                 —C(O)—NY¹Y² or —C(O)—OY³,             -   where Y¹ and Y² independently of one another represent                 —H, —NH₂ or —(CH₂)₀₋₃Z′,             -   where Y³ represents —H or —(CH₂)₀₋₃Z′,             -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   R⁶ and R⁷ independently of one another represent —H, cyano,             (optionally fluorinated) C₁₋₁₀-alkyl, (optionally             fluorinated) C₂₋₁₀-alkenyl, (optionally fluorinated)             C₂₋₁₀-alkynyl, hydroxy, —NO₂, —NH₂, —COOH or halogen (in             particular —F, —Cl, —Br),         -   R⁸ represents (optionally fluorinated) C₁₋₁₀-alkyl,             (optionally fluorinated) C₂₋₁₀-alkenyl, (optionally             fluorinated) C₂₋₁₀-alkynyl, (optionally fluorinated)             C₄₋₁₀-cycloalkyl or —(CH₂)₀₋₂—(HZ²),             -   where HZ² represents a 4- to 7-membered heterocycle                 having up to two heteroatoms selected from the group                 consisting of N, O and S, where each of these groups may                 be substituted by —OH, —COOH or —NH₂ or -L-#1;             -   where -L-#1 represents the linker and #1 represents the                 bond to the binder or derivative thereof,                 -   and             -   where -MOD as defined supra represents                 —(NR¹⁰)n-(G1)o-G2-G3,             -   where R¹⁰ represents H or C₁-C₃-alkyl;             -   where G1 represents NH—C(═O)—, —C(═O)—NH— or

-   -   -   -    (where, if G1 represents —NHC(═O)— or

-   -   -   -    R¹⁰ does not represent —NH₂);             -   where n is 0 or 1;             -   where o is 0 or 1;             -   where G2 represents a linear and/or branched hydrocarbon                 group which has 1 to 10 carbon atoms and which may be                 interrupted once or more than once by one or more of the                 groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—,                 —NR^(y)C(═O)—, —C(═O)—NR^(y)—, —NR^(y)NR^(y)—,                 —S(═O)₂—NRyNRy-, —C(═O)—NR^(y)NR^(y)—, —C(═O)—,                 —CR^(x)═N—O—, and where the hydrocarbon chain including                 any side chains may be substituted by —NH—C(═O)—NH₂,                 —COOH, —OH, —NH₂, NH—CNNH₂, sulphonamide, sulphone,                 sulphoxide or sulphonic acid,             -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,                 C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be                 substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂,                 NH—CNNH₂, sulphonamide, sulphone, sulphoxide or                 sulphonic acid,             -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,             -   where G3 represents —H or COOH, and             -   where -MOD preferably has at least one —COOH group;

        -   and the salts, solvates, salts of the solvates, and epimers             thereof.

The conjugates according to the invention can have chemically labile linkers, enzymatically labile linkers or stable linkers. Particular preference is given to stable linkers and linkers which can be cleaved by legumain or cathepsin.

The invention furthermore provides processes for preparing the site specific homogeneous conjugates according to the invention, and also precursors and intermediates for the preparation.

The preparation of the conjugates according to the invention regularly comprises the following steps:

-   -   (i) Preparation of a linker precursor which optionally carries         protective groups and has a reactive group which is capable of         coupling to the binder;     -   (ii) Conjugation of the linker precursor to the derivative,         which optionally carries protective groups, of a low-molecular         weight KSP inhibitor (preferably a KSP inhibitor having the         substructure I(sub), particularly preferably of formula (Ia) and         in particular of formula (IIa), where in these formulae there is         as yet no bond to a linker), giving a reactive KSP         inhibitor/linker conjugate which optionally carries protective         groups;     -   (iii) Removal of any protective groups present in the KSP         inhibitor/linker conjugate and     -   (iv) Site specific conjugation of the binder to the KSP         inhibitor/linker conjugate, preferably using transglutaminase,         giving the binder/KSP inhibitor site specific homogenous         conjugate according to the invention.

Attachment of the reactive group may also take place after the construction of an optionally protected KSP inhibitor/linker precursor conjugate.

As illustrated above, conjugation of the linker precursor to a low-molecular weight KSP inhibitor may take place by substitution of a hydrogen atom at R^(1a), R^(2a), R^(4a) or R¹⁰ in substructure I(sub), R^(1a), R^(2a), R^(3a), R^(4a), R^(8a) or R¹⁰ in formula (Ia), or R¹, R², R³, R⁴, R⁵, R⁸ or R¹⁰ in formula (IIa) by the linker. In the synthesis steps prior to the conjugation, any functional groups present may also be present in protected form. Prior to the conjugation step, these protective groups are removed by known methods of peptide chemistry. Conjugation can take place chemically by various routes, as shown in an exemplary manner in Schemes 2 to 6 in the examples. In particular, it is optionally possible to modify the low-molecular weight KSP inhibitor for conjugation to the linker, for example by introduction of protective groups or leaving groups to facilitate substitution.

In particular, the invention provides low-molecular weight KSP inhibitors conjugated to a binder. These r binder conjugates have the following general formula (IIIa):

-   -   where     -   X₁ represents N, X₂ represents N and X₃ represents C; or     -   X₁ represents N, X₂ represents C and X₃ represents N; or     -   X₁ represents CH or CF, X₂ represents C and X₃ represents N; or     -   X₁ represents NH, X₂ represents C and X₃ represents C; or     -   X₁ represents CH, X₂ represents N and X₃ represents C     -   (with X₁ representing CH, X₂ representing C and X₃ representing         N being preferred);     -   R¹ represents —H, -L-BINDER, MOD or —(CH₂)₀₋₃Z,         -   where Z represents —H, —NHY³, —OY³, —SY³, halogen,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or             —CH(CH₂W)Z′, and         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—(C═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R² represents -L-BINDER, H, -MOD, —C(═O)—CHY⁴—NHY⁵ or         —(CH₂)₀₋₃Z, or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents L-#1, —H, —NH₂, —SO₃H, —COOH, —SH, or             —OH;         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R⁴ represents -L-BINDER, —H, —C(O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R⁴ represents a group of         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—, or         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)—, or the         cathepsin cleavable group or formula         R²¹—(C═O)₍₀₋₁₎—(P3)⁽¹⁻²⁾-P2-,         -   where R²¹ represents —H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group,             which may be substituted one or more times with —NH₂,             —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,             —N(alkyl)-C(═O)-alkyl, —SO₃H, —SO₂NH₂, —S(═O)₂—N(alkyl)₂,             —COOH, —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or OH, or is —H or a             group —O_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1,         -   where v is a number from 1 to 20, and         -   where R²² represents —H, -alkyl (preferably C1-12-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂;         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid independently selected from Gly,             Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr,             Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or one             of the corresponding N-alkyl amino acids, preferably             N-methyl amino acids;     -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents L-#1, —H, —NH₂, —SO₃H, —COOH, —SH,             halogen C₁₋₄ Alkyl, C₁₋₄ Haloalkyl, C₁₋₄ Alkoxy,             Hydroxyl-substituted C₁₋₄ Alkyl, COO(C₁₋₄ Alkyl) or —OH; and             wherein the hydrogen atom of the secondary amino group in             the pyrrolidine ring may be replaced by             R²¹—C(═O)—P3₍₀₋₂₎-P2-NH—CH(CH₂C(═O)NH₂)—C(═O)—SIG-;         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid independently selected from Gly,             Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr,             Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or one             of the corresponding N-alkyl amino acids, preferably             N-methyl amino acids;         -   where -SIG is a self-immolative group, which, upon cleavage             of the C(═O)—SIG bond provides the free secondary amine;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         C(═N—NH₂)—;     -   R³ represents -L-BINDER, -MOD or an optionally substituted         alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl,         heterocycloalkyl group, preferably -L-BINDER, or a C₁₋₁₀-alkyl,         C₆₋₁₀-aryl or C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl,         C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or C₅₋₁₀-heterocycloalkyl group which         may be substituted by 1-3 —OH groups, 1-3 halogen atoms, 1-3         halogenated alkyl groups (each having 1-3 halogen atoms), 1-3         —O-alkyl groups, 1-3 —SH groups, 1-3 —S-alkyl groups, 1-3         —O—C(═O)-alkyl groups, 1-3 —O—C(═O)—NH-alkyl groups, 1-3         —NH—C(═O)-alkyl groups, 1-3 —NH—C(═O)—NH-alkyl groups, 1-3         —S(═O)_(n)-alkyl groups, 1-3 —S(═O)₂—NH-alkyl groups, 1-3         —NH-alkyl groups, 1-3 —N(alkyl)₂ groups, 1-3 —NH₂ groups or 1-3         —(CH₂)₀₋₃Z groups,         -   where n is 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH         -   where “alkyl” preferably represents C₁₋₁₀-alkyl);     -   R⁵ represents -L-BINDER, —H, —NH₂, —NO₂, halogen (in particular         F, Cl, Br), —CN, —CF₃, —OCF₃, —CH₂F, —CH₂F, —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         C₄₋₁₀-cycloalkyl, fluoro-C₄₋₁₀-cycloalkyl or —(CH₂)₀₋₂—(HZ²),         -   where HZ² represents a 4- to 7-membered heterocycle having             up to two heteroatoms selected from the group consisting of             N, O and S (preferably oxetane), where each of these groups             may be substituted by —OH, —COOH or             -   —NH₂ or -L-BINDER;         -   where L represents a linker and         -   BINDER represents a binder or a derivative thereof, where             the binder may optionally be attached to a plurality of             active compound molecules,         -   where one representative of R¹, R², R³ R⁴, R⁵, R⁸ and R¹⁰             represents -L-binder;     -   R⁶ and R⁷ independently of one another represent —H, cyano,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy, —NO₂, —NH₂, —COOH or halogen (in particular —F, —Cl,         —Br),     -   -MOD represents —(NR¹⁰)n-(G1)o-G2-G3,         -   where R¹⁰ represents —H or C₁-C₃-alkyl;         -   where G1 represents NH—C(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents NH—C(═O)— or

-   -   -    R¹⁰ does not represent NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1; and         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,             —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—,             —C(═O)—NR^(y)NR^(y)—,         -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid, or             represents —C(═O)—, —CR^(x)═N—O—         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,         -   where the hydrocarbon chain including any side chains may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   where G3 represents —H or —COOH, and         -   where -MOD preferably has at least one group —COOH;

    -   and the salts, solvates, salts of the solvates, and epimers         thereof.

DESCRIPTION OF THE FIGURES

FIG. 1 shows alignment of the TWEAKR cysteine-rich domain (amino acids 34 to 68) of various species. (The numbers show the amino acid position in full-length constructs including the signal sequences; “Human” shows amino acids 34 to 68 of SEQ ID NO: 169, “Rat” shows amino acids 7 to 41 of SEQ ID NO: 134, “Mac” shows amino acids 7 to 41 of SEQ ID NO: 133, “Pig” shows amino acids 7 to 41 of SEQ ID NO: 135, “Mouse” shows amino acids 7 to 41 of SEQ ID NO: 137, “Dog” shows amino acids 7 to 41 of SEQ ID NO: 136).

FIG. 2A shows a schematic diagram of the structure of TWEAKR (SEQ ID NO: 169). The diagram shows the extracellular domain (amino acids 28-80) (SEQ ID NO: 168) including the cysteine-rich domain (36-67), the transmembrane domain—TM (81-101) and the intracellular domain (102-129). TPP-2202 the complete ectodomain (28-80), fused to the Fc domain of hIgG1. TPP-2203 extracellular domain with N- and C-terminal truncation (34-68), fused to the Fc domain of hIgG1. Disulphide bridges Cys36-Cys49, Cys52-Cys67 and Cys55-Cys64 are indicated by black bars. N-terminally and C-terminally, TPP-2203 contains two amino acids more and one amino acid more, respectively, than the unmodified cysteine-rich domain to ensure proper folding. TPP-1984 extracellular domain having C-terminal truncation (28-68), fused to an HIS6 tag. All three constructs show comparable binding to the antibodies according to the invention and PDL-192 (TPP-1104). P4A8 (TPP-1324) binds only to the full-length extracellular domain (TPP-2202).

FIG. 2B shows an amino acid sequence of the extracellular domain (SEQ ID NO: 168): It has been published that the amino acid 64 is essential for TWEAK ligand binding; and the amino acid 47 is essential for binding of the antibodies according to the invention, as was determined here.

FIG. 3 shows the interaction of the TWEAKR ectodomain with antibodies and reference antibodies. What is shown is the result of an ELISA with TWEAKR-Fc fusion protein coating (TPP-2202, 1 μg/ml) and with 0.08 μg/ml (open bars) and 0.03μ/ml (solid bars) of biotinylated IgG as soluble binding partner. Detection was carried out using streptavidin-HRP and Amplex Red substrate. Y is the “ELISA signal intensity [Rfu]”; X are the “tested antibody constructs”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192(TPP-1104)”; d is “P4A8(TPP-1324)”; e is “P3G5(TPP-2195)”; f is “136.1(TPP-2194)”; h is “ITEM1”; i is “ITEM4”; j is a mouse isotype control; k is a human isotype control. All antibodies examined show saturated binding at a concentration of 80 ng/ml.

FIG. 4 shows the interaction of the cysteine-rich domain of TWEAKR with antibodies according to the invention and reference antibodies. What is shown is the result of an ELISA with TWEAKR (34-68)-Fc fusion protein coating (TPP-2203, 1 μg/ml) and 0.08 μg/ml (open bars) and 0.3 μ/ml (solid bars) of biotinylated IgG as soluble binding partner. Detection was carried out using streptavidin-HRP and Amplex Red substrate. X are the “antibody constructs tested”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192(TPP-1104)”; d is “P4A8(TPP-1324)”; e is “P3G5(TPP-2195)”; f is “136.1(TPP-2194)”; h is “ITEM1”; i is “ITEM4”; j is a mouse isotype control; k is a human isotype control. All antibodies examined show saturated binding at a concentration of 80 ng/ml.

FIG. 5 shows the interaction of TWEAKR (28-68) with antibodies according to the invention and reference antibodies. What is shown is the result of an ELISA with TWEAKR (28-68)-HIS coating (TTP-1984, 1 μg/ml) and 0.08 μg/ml (open bars) and 0.3 μ/ml (solid bars) of biotinylated IgG as soluble binding partner. Detection was carried out using streptavidin-HRP and Amplex Red substrate. X are the “antibody constructs tested”: a is “TPP-2090”; b is “TPP-2084”; c is “PDL-192(TPP-1104)”; d is “P4A8(TPP-1324)”; e is “P3G5(TPP-2195)”; f is “136.1(TPP-2194)”; his “ITEM1”; i is “ITEM4”; j is a mouse isotype control; k is a human isotype control. All antibodies examined show saturated binding at a concentration of 80 ng/ml.

FIG. 6A shows an alanine scan of the cysteine-rich domain. Muteins of TWEAKR(34-68)-Fc were analysed for PDL-192(TPP-1104) (X)- and TPP-2090 (Y)-binding. S37A, R38A, S40A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and L65A muteins were expressed in HEK293 cells (black diamonds). PFL192(TPP-1104) and TPP-2090 were coated (1 μg/ml) and an 8-fold diluted supernatant of the HEK293 fermentation broth was added for TWEAKR protein binding X is the “ELISA intensity of the PDL-192/TTP-1104) interaction [Rfu]”, Y is the “ELISA intensity of the TPP-2090 interaction [Rfu]”. TPP-2090 (Y) shows reduced binding for the D74A-TWEAKR mutein (closed box), and PDL-192(TPP-1104) (X) shows reduced binding to R56A (spotted box).

FIG. 6B shows the correlation blots for the 8-fold diluted supernatants of the TWEAKR expression broth, with PDL-192(TPP-1104) on the X axis and TPP-2090 on the Y axis. Y is the “binding in % normalized to the wild-type binding signal [%]”, 1 is “TPP-2090”; 2 is “PDL-192(TPP-1104)”; 3 is “P4A8(TPP-1324)”. (1 μg/ml), the TWEAKR variant was added at 250 ng/ml, detection was via anti-HIS HRP. Compared to the wild-type construct, TTP-2090 shows less than 5% binding.

FIG. 7 shows the NMR structure of the TWEAKR ectodomain as published by Pellegrini et al. (FEBS 280:1818-1829). TWEAK binding depends on L46 (Pellegrini et al.), TTP-2090 binding depends on D47 and PDL-192 binds to R56. PDL-192 binds opposite the TWEAK ligand binding site, TPP-2090 binds directly to the TWEAK ligand site.

FIG. 8 depicts the strategy using transglutaminase catalyzed conjugation of aglycosylated antibodies.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides site specific homogeneous conjugates of a binder or derivative thereof with one or more active compound molecules, the active compound molecule being a kinesin spindle protein inhibitor (KSP inhibitor) attached to the binder via a linker L and the linker L being conjugated to a specific site at the binder, preferably a glutamine side chain of the binder.

More specifically the invention provides site specific homogeneous conjugates of a binder or derivative thereof with one or more active compound molecules, the active compound molecule being a kinesin spindle protein inhibitor (KSP inhibitor) attached to the binder via a linker L and the linker L being conjugated to a glutamine side chain of the binder using transglutaminases (TGases).

The conjugate according to the invention can be represented by the general formula BINDER

L

KSP]_(m)]_(n) where BINDER represents the binder, preferably an antibody, L represents the linker, KSP represents the KSP inhibitor, m represents a number from 1 to 5, preferably 1, and n represents 2 to 10, preferably 2 to 4, and also preferably 2 or 4. Here, m is the number of KSP inhibitors per linker and n the number of KSP inhibitor/linker conjugates per BINDER. The sum of all KSP present in the conjugate is thus the product of m and n. KSP-L preferably has the formula (IIa) shown above. The binder is preferably a binder peptide or protein such as, for example, an antibody. If the binder is an antibody it comprises an acceptor glutamine, preferentially in the constant region. Such acceptor glutamines can be introduced by mutations of suitable positions into glutamine (e.g. mutation N297Q, Kabat EU numbering) or by generation of deglycosylated or aglycosylated antibodies (e.g. by enzymatic deglycosylation by PNGase F or by mutation of N297X, Kabat EU numbering). In that later case of a deglycosylated or an aglycosylated antibody the glutamine Q295 (Kabat EU numbering) becomes an acceptor glutamine. Highly preferred is an antibody comprising a mutation N297A or N297Q (Kabat EU numbering). Furthermore, the linker is attached to glutamine residues of the binder peptide or protein or derivative thereof. Particular preference is given to binding to glutamine residues of an antibody.

Binders which can be used according to the invention, KSP inhibitors which can be used according to the invention and linkers which can be used according to the invention which can be used in combination without any limitation are described below. In particular, the binders represented in each case as preferred or particularly preferred can be employed in combination with the KSP inhibitors represented in each case as preferred or particularly preferred, optionally in combination with the linkers represented in each case as preferred or particularly preferred.

KSP Inhibitors and their Binder Conjugates

Low-molecular weight KSP inhibitors are known, for example, from WO2006/044825; WO2006/002236; WO2005/051922; WO2006/060737; WO03/060064; WO03/040979; and WO03/049527.

As a rule, KSP inhibitors have the following substructure I(sub):

-   -   where     -   #a represents a bond to the rest of the molecule;     -   R^(1a) represents —H or —(CH₂)₀₋₃Z,         -   where Z represents —H, halogen, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or             —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R^(2a) represents —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R^(4a) represents —H, —C(O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R^(4a) represents a group of         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or the         cathepsin cleavable group or formula         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-,         -   where R²¹ represents —H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group,             which may be substituted one or more times with —NH₂,             —NH-alkyl, —N(alkyl)₂, NH—C(═O)-alkyl, N(alkyl)-C(═O)alkyl,             —SO₃H, —SO₂NH₂, —S(═O)₂—N(alkyl)₂, —COOH, —C(═O)—NH₂,             —C(═O)—N(alkyl)₂, or OH; or which represents —H or a group             —O_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1,         -   where v is a number from 1 to 20,         -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂;         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His or one of the             corresponding N-alkyl amino acids, preferably N-methyl amino             acids;     -   or     -   R^(2a) and R^(4a) together represent (with formation of a         pyrrolidine ring) CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, halogen (preferably —F or —Cl),             —NH₂, —COOH,         -   —SO₃H, —SH, C₁₋₄-alkyl, halo-C₁₋₄alkyl, C₁₋₄-alkoxy, hydroxy             substituted C₁₋₄-alkyl, —C(═O)—O—(C₁₋₄-alkyl) or —OH.

Particularly frequently encountered is the following substructure II(sub)

-   where #a, R^(1a), R^(2a) and R^(4a) have the same meaning as in     I(sub) and R^(12a) and R^(13a) represents —H or -   R^(12a) and R^(13a) together (with formation of a piperidine ring)     represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,     -   where R¹⁰ represents —H, —NH₂, —COOH, —SO₃H, —SH or —OH;     -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or         —C(═O)—OY³,     -   where Y¹ and Y² independently of one another represent —H, —NH₂         or —(CH₂CH₂O)₀₋₃—(CH₂)₀₃Z′         -   (e.g. —(CH₂)₀₋₃Z′),     -   where Y³ represents —H or —(CH₂)₀₋₃Z′,     -   where Z′ represents —H, —SO₃H, —NH₂, —COOH or         —(C═O)—NH—CHY⁴)₁₋₃COOH,     -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is         optionally substituted by —NH—C(═O)—NH₂, or represents aryl or         benzyl which are optionally substituted by —NH₂.

In particular, a number of KSP inhibitors have the substructure II(sub) where R^(1a), R^(2a), R^(4a), R^(12a) and R^(13a) represent H.

According to the invention, use may be made of KSP inhibitors of the substructure I(sub) or the substructure II(sub). The KSP inhibitors which are used in accordance with the invention also include, for example, ispinesib (Cytokinetics/GSK), MK-0731 (Merck), AZD4877 (AstraZeneca), ARRY-520 (Array BioPharma) and ARQ 621 (ArQule).

KSP inhibitors which are preferred in accordance with the invention have the following basic structure:

-   -   where     -   R^(1a) represents —H, -MOD or —(CH₂)₀₋₃Z,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R^(2a) represents —H, MOD, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R^(4a) represents —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R^(4a) represents a group of         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂—(C═O)—NH₂)—C(═O)— or         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)— or the         cathepsin cleavable group or formula         R²¹—(C═O)₍₀₋₁₎—(P3)⁽¹⁻²⁾-P2-,         -   where R²¹ represents —H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group,             which may be substituted one or more times with —NH₂,             —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,             —N(alkyl)-C(═O)-alkyl, —SO₃H, —SO₂NH₂, —S(═O)₂—N(alkyl)₂,             —COOH, —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or —OH;             -   or represents —H or a group —O_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1         -   where v s a number from 1 to 20,         -   where R²² represents —H, -alkyl (preferably C1-12-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂);         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His or one of the             corresponding N-alkyl amino acids, preferably N-methyl amino             acids;     -   or     -   R^(2a) and R^(4a) together represent (with formation of a         pyrrolidine ring) —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents H, halogen (preferably F), —NH₂, —COOH,             —SO₃H, —SH or —OH, and wherein the hydrogen atom of the             secondary amino group in the pyrrolidine ring may be             replaced by             R²¹—C(═O)—P3₍₀₋₂₎-P2-NH—CH(CH₂C(═O)NH₂)—C(═O)—SIG-;         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His or one of the             corresponding N-alkyl amino acids, preferably N-methyl amino             acids;         -   where -SIG is a self-immolative group, which, upon cleavage             of the C(═O)—SIG bond provides the free secondary amine;     -   R^(3a) represents MOD or an optionally substituted alkyl,         cycloalkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl         group, preferably a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or C₆₋₁₀-aralkyl,         C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where -MOD is represented as defined infra,         -   where n is 0, 1 or 2,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NHC(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH     -   R^(8a) represents C₁₋₁₀-alkyl;     -   HZ represents a mono- or bicyclic heterocycle which may be         substituted by one or more substituents selected from the group         consisting of halogen, C₁₋₁₀-alkyl groups, C₆₋₁₀-aryl groups and         C₆₋₁₀-aralkyl groups which may optionally be substituted by         halogen;     -   -MOD as defined supra represents (NR¹⁰)n-(G1)o-G2-G3,         -   where R¹⁰ represents —H; halogen or C₁-C₃-alkyl;         -   where G1 represents NH—C(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents NHC(═O)— or

-   -   -    R¹⁰ does not represent NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1;         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups             -   —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,                 —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NRyNRy-,                 —C(═O)—NR^(y)NR^(y)—, —C(═O)—, —CR^(x)═N—O—,             -   and where the hydrocarbon group including any side                 chains may be substituted by —NHC(═O)—NH₂, —COOH, —OH,                 —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or                 sulphonic acid,         -   where R^(y) represents H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NHC(═O)—NH₂, —COOH, —OH, —NH₂, NH—CNNH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl),         -   where G3 represents —H or —COOH,         -   where -MOD preferably has at least one group —COOH;

    -   and the salts, solvates, salts of the solvates, and epimers         thereof.

According to the invention, such a kinesin spindle protein inhibitor can be attached to the linker by substitution of a hydrogen atom at R^(1a), R^(2a), R^(3a), R^(4a), R^(8a) or R¹⁰ or optionally via one of the substituents of HZ, in particular via R^(1a), R^(2a), R^(3a), R^(4a) or R¹⁰.

The substituents of the formula (Ia) preferably have the following meanings, where these preferred meanings are preferably combined with one another:

-   R^(1a) preferably represents H or —(CH₂)₀₋₃Z,     -   where Z represents —H, halogen, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y²         or —C(═O)—OY³,     -   where Y¹ and Y² independently of one another represent —H, —NH₂,         —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′,     -   where Y³ represents —H or —(CH₂)₀₋₃Z′,     -   where Z′ represents —H, —SO₃H, —NH₂, —COOH,         —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,     -   where W represents —H or —OH,     -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is         optionally substituted by —NH—C(═O)—NH₂, or represents aryl or         benzyl which are optionally substituted by —NH₂; -   R^(2a) and R^(4a) independently of one another preferably represent     —H, -L-#1, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z, or R^(2a) and R^(4a)     together (with formation of a pyrrolidine ring) represent CH₂—CHR¹⁰—     or —CHR¹⁰—CH²—,     -   where R¹⁰ represents —H, —SO₃H, —NH₂, —COOH, —SH or —OH,     -   where Z represents —H, halogen, —OY³, —SY³, NHY³, —C(═O)—NY¹Y²         or —C(═O)—OY³,     -   where Y¹ and Y² independently of one another represent —H, —NH₂         or —(CH²)₀₋₃Z′,     -   where Y³ represents —H or —(CH₂)₀₋₃Z′,     -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH,     -   where Y⁴ independently of one another represents linear or         branched C₁₋₆-alkyl which is optionally substituted by         —NH—C(═O)—NH₂, or represents aryl or benzyl which are optionally         substituted by —NH₂,     -   where Y⁵ represents H or —C(═O)—CHY⁶—NH₂,     -   where Y⁶ represents linear or branched C₁₋₆-alkyl.

Alternatively, R^(4a) may be R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)— or a cathepsin cleavable group of formula R²¹—(C═O)₍₀₋₁₎—(P3)₍₁₋₂₎-P2-. In another alternative, the hydrogen atom of —NH on the pyrrolidine ring is replaced by R²¹—C(═O)—P3-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—SIG-.

The group R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—, R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)— or R²¹—C(═O)—P3-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—SIG- are believed to be cleaved by the enzyme legumain in vivo. In the following, these groups will thus be referred to as “legumain cleavable groups”. The legumain cleavable group has the formula —(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or —(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)—. In the conjugates of the present invention, this group has preferably the formula R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)#, i.e. the legumain cleavable group has a group R²¹ at one end, and at the other end (−#) it binds to the amino group corresponding position R^(4a) in Formula Ia.

—NH—CH(CH₂C(═O)—NH₂)—C(═O)— (i.e. asparagine) and —NH—CH(CH₂COOH)—C(═O)— (i.e. aspartic acid) are present in the natural L-configuration. The legumain cleavable group contains, in addition to asparagine, 1 to 3 additional amino acids (—P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—; —P3-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—; —(P3)₍₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—), thus is a di-, tri-, or tetrapeptide or derivative thereof (dipeptide: —P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—; tripeptide: —P3-P2-NH—CH(CH₂C(═O)NH₂)—C(═O)—; tetrapeptide: —(P3)₂-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—). The same applies in case the legumain cleavable group contains aspartic acid.

P2 is an amino acid selected from Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline und His, preferably selected from Ala, Gly, Val, Leu, Ile, Pro, Ser, Thr, citrulline und Asn. P2 is generally present in the natural L-configuration. Particularly preferred is L-Ala.

P3 is an amino acid independently selected from Gly, Pro, Ala, Val, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline, or its corresponding N-alkyl-amino acids, preferably N-methyl-amino acids. P3 is preferably selected from His, Pro, Ala, Val, Leu, Ile, Gly, Ser, citrulline und Gln. P3 is generally present in the natural L-configuration vor. Particularly preferred is L-Ala.

A particularly preferred legumain cleavable group is -L-Ala-L-Ala-L-Asn- (e.g. R²¹-L-Ala-L-Ala-L-Asn-#).

-   R21 preferably represents —H, C₁₋₅-alkyl, C₅₋₁₀-aralkyl,     C₁₋₅-alkoxy, C₆₋₁₀-aryloxy, C₅₋₁₀-heteroalkyl-,     C₅₋₁₀-heterocycloalkyl-, heteroaryl-, heteroaryl-alkyl-,     C₅₋₁₀-heteroalkoxy-, or a C₅₋₁₀-heterocycloalkoxy group, which each     may be substituted one or more times with —COOH, —C(═O)—OAlkyl,     —C(═O)—O—NH₂, —NH₂ or —N(Alkyl)₂); or represents a group     —O_(x)—(CH₂CH₂O)_(y)—R²²,     -   where x is 0 or 1     -   where v is a number from 1 to 10, and     -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),         —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂.

“Alkyl” here means an alkyl group with up to 20 carbon atoms, preferably C₁₋₁₂-alkyl.

The cathepsin cleavable group has the formula —(C═O)₍₀₋₁₎—(P3)₍₁₋₂₎-P2-. In the conjugates of the present invention, this group has preferably the formula R²¹—(C═O)₍₀₋₁₎—(P3)₍₁₋₂₎-P2-#, i.e. the cathepsin cleavable group has a group R²¹ at one end, and at the other end (−#) it binds to the amino group corresponding position R^(4a) in Formula Ia. Here R²¹, P2 and P3 have the same meaning as in the legumain cleavable group. Particularly preferred cathepsin cleavable groups are those in which P2 is selected from alanine, lysine und citrulline, und P3 is selected from valine, alanine und phenylalanine, particularly those of formula R²¹—(C═O)₍₀₋₁₎—P3-P2-.

-   R³ preferably represents an optionally substituted alkyl, aryl,     heteroaryl, heteroalkyl, heterocycloalkyl group, -L-#1, -MOD, or a     C₁₋₁₀-alkyl, C₆₋₁₀-aryl or C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl,     C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or C₅₋₁₀-heterocycloalkyl group which may     be substituted by 1-3 —OH groups, 1-3 halogen atoms, 1-3 halogenated     alkyl groups (each having 1-3 halogen atoms), 1-3 O-alkyl groups,     1-3 —SH groups, 1-3 —S-alkyl groups, 1-3 —O—C(O)-alkyl groups, 1-3     —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3     —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3     —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂     groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,     -   where n is 0, 1 or 2,     -   where Z represents —H, halogen, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y²         or         -   —C(═O)—OY³,     -   where Y¹ and Y² independently of one another represent —H, —NH₂         or —(—CH₂)₀₋₃Z′     -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,         -   —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,     -   where Z′ represents —H, —SO₃H, —NH₂ or COOH,     -   where “alkyl” preferably represents Chm-alkyl. -   R^(8a) preferably represents C₁₋₁₀-alkyl.

HZ preferably represents a mono- or bicyclic heterocycle which may be substituted by one or more substituents selected from the group consisting of halogen, C₁₋₁₀-alkyl groups, C₆₋₁₀-aryl groups and C₆₋₁₀-aralkyl groups which may optionally be substituted by halogen.

C₁₋₁₀-Alkyl in the context of the invention (i.e. in the formula above and also in the formulae that follow) represents a linear or branched alkyl radical having 1 to 10 carbon atoms. Examples which may be mentioned as being preferred are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl.

C₆₋₁₀-Aryl- in the context of the invention represents a mono- or bicyclic aromatic homocycle, for example phenyl and naphthyl.

C₆₋₁₀-Aralkyl group in the context of the invention represents a monocyclic aromatic homocycle, by way of example phenyl, to which a C1-C4-alkyl group is attached. An exemplary C₆₋₁₀-aralkyl group is benzyl.

C₅₋₁₀-Heteroaryl in the context of the invention represents a mono- or bicyclic aromatic heterocycle having a total of 6 to 10 ring atoms, where the ring(s) contains/contain one or two ring heteroatoms from the group consisting of N, O, S, SO and SO₂ and which is attached via a ring carbon atom or optionally a ring nitrogen atom. Examples which may be mentioned are pyridyl, furanyl, pyrimidyl, imidazolyl, thienyl, thiophenyl, isoxazoyl, isothiazoyl, 1,2,3-oxadiazoyl, furazanyl, 1,2,3-triazoyl, 1,2,4-triazoyl, pyridazyl, pyrrolyl, triazinyl, indolyl, quinolinyl, quinazolinyl, 1,3-benzodioxol, isoindolyl, indazolyl, 1H-pyrazolo[3,4-d]pyrimidyl, benzotriazolyl, isoquinolinyl, cinolinyl, phthalazinyl, pteridinyl, naphthyridinyl, benzimidazolinyl, benzothiazolinyl, benzoxazolinyl, 3,4-methylenedioxyphenyl and benzo[6]furanyl.

Mono- or bicyclic heterocycle in the context of the invention represents a mono- or bicyclic heterocycle having a total of 5 to 10 ring carbon atoms, where the ring(s) contains/contain one to three ring heteroatoms from the group consisting of N, O, S, SO and SO₂ and which is attached via a ring carbon atom or optionally a ring nitrogen atom. Examples which may be mentioned are piperidyl, pyrrolinyl, morpholinyl, 3,4-methylenedioxyphenyl and tetrahydrofuranyl.

Halogen atom in the context of the invention represents F, Cl, Br or I.

As used herein, the term “heteroalkyl” refers to a straight or branched alkyl group that contains one or more heteroatoms, that is, an element other than carbon (including but not limited to oxygen, sulfur, nitrogen, phosphorus) in place of one or more carbon atoms.

Whenever a group is described as being “substituted” that group substituted with one or more of the indicated substituents. If no substituents are indicated, it is meant that the indicated “substituted” group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamyl, thiocarbamyl, amido, sulfonamido, sulfonamido, carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, an amino, a mono-substituted amino group and a di-substituted amino group, and protected derivatives thereof.

Where the number of substituents is not specified (e.g. haloalkyl), there may be one or more substituents present. For example “haloalkyl” may include one or more of the same or different halogens. As another example, “C˜—C˜ alkoxyphenyl” may include one or more of the same or different alkoxy groups containing one, two or three atoms.

By substitution of a hydrogen atom at R^(1a), R^(2a), R^(4a) or R¹⁰ in substructure I(sub) or substructure II(sub), or R^(1a), R^(2a), R^(3a), R^(4a), R^(8a) or R¹⁰ at HZ in formula (Ia), the compound of the formula (Ia) may be attached to a linker in a manner known to the person of average skill. Particularly preferably, the substitution of the hydrogen atom takes place at R^(1a), R^(2a), R^(3a), R^(4a) or at the pyrrolidine ring formed by R^(2a) and R^(4a). This conjugation can take place chemically by various routes, as shown in an exemplary manner in Schemes 2 to 6 in the examples. In particular, it is optionally possible to modify the low-molecular weight KSP inhibitor for the conjugation to the linker, for example by introducing protective groups or leaving groups to facilitate substitution (such that in the reaction said leaving group, and not a hydrogen atom, is substituted by the linker). The KSP inhibitor-linker molecules obtained in this manner (where the linker has a reactive group for coupling to the binder) can then be reacted with the binder to give a binder conjugate according to the invention. In the experimental section, this procedure is illustrated in an exemplary manner by some examples.

Preferred for R^(1a) are —H, —COOH, —C(═O)—NHNH₂, —(CH₂)₁₋₃NH₂, —C(═O)—NZ″(CH₂)₁₋₃—NH₂ and —C(═O)—NZ″CH₂COOH,

where Z″ represents —H or —NH₂.

Preferred for R^(2a) and R^(4a) are H, or R^(2a) and R^(4a) together (with formation of a pyrrolidine ring) represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,

where R¹⁰ represents —H.

Preferred for R^(3a) is C₁₋₁₀-alkyl-, which may be substituted by OH, —O-alkyl, —SH, —S-alkyl, —O—C(═O)-alkyl, —O—C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —NH—C(═O)—NH-alkyl, —S(═O)_(n)-alkyl, —S(═O)₂—NH-alkyl, —NH-alkyl, —N(alkyl)₂ or —NH₂,

where alkyl is preferably C₁₋₃-alkyl and

n is 0, 1 or 2.

Preferred for R^(8a) is a branched C₁₋₅-alkyl group, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl and tert-butyl.

Preferred for HZ is a mono- or bicyclic heterocycle which may be substituted by one or more substituents selected from the group consisting of halogen, C₁₋₁₀-alkyl groups, C₆₋₁₀-aryl groups and C₆₋₁₀-aralkyl groups which may optionally be substituted by halogen.

Particularly preferably, HZ is a substituted pyrrole, pyrazole, imidazole, quinazoline or dihydroquinazoline which is substituted in the ortho-position (with respect to the substituents with R1a etc.) by an optionally substituted benzyl group. Furthermore, the substituted pyrrole, pyrazole, imidazole or quinazoline can preferably be substituted by oxo (in the case of dihydroquinazoline) or a phenyl group substituted by 1 or 2 halogen atoms. Particularly preferably, HZ is a substituted pyrrole.

A KSP inhibitor which is preferably used is ispinesib. A further preferred KSP inhibitor is Arry-520.

Other particularly preferred compounds of the structure KSP-L- have the formula (IIa) or (II) below:

-   -   where     -   X₁ represents N, X₂ represents N and X₃ represents C; or     -   X₁ represents N, X₂ represents C and X₃ represents N; or     -   X₁ represents CH or CF, X₂ represents C and X₃ represents N; or     -   X₁ represents NH, X₂ represents C and X₃ represents C; or     -   X₁ represents CH or CF, X₂ represents N and X₃ represents C;     -   (with X₁ representing CH, X₂ representing C and X₃ representing         N being preferred);     -   R¹ represents —H, -L-#1, -MOD or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NHC(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂;     -   R² represents —H, -L-#1, -MOD, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′, and         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NHC(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R⁴ represents —H, -L-#1, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NHC(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂, and         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R⁴ represents a group of     -   R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)— or the         cathepsin cleavable group R²¹—(C═O)₍₀₋₁₎—(P3)₍₁₋₂₎-P2-,         -   where R²¹ represents H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             GAD-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group             (which may be substituted one or more times with NH₂,             —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,             N(alkyl)-C(═O)-alkyl, —SO₃H,             -   —SO₂NH₂, —S(O)₂—N(alkyl)₂, —COOH,             -   —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or —OH), —H or a group                 —(O)_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1 and         -   where v is a number from 1 to 20,         -   where R²² represents —H, -alkyl (preferably Chu-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂);         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid independently selected from Gly,             Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr,             Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or one             of the corresponding N-alkyl amino acids, preferably             N-methyl amino acids; in case there is more than one amino             acid P3, P3 may have the same or different amino acids, as             defined above;         -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, halogen (preferably —F), —NH₂,             —SO₃H, —COOH, —SH or —OH, and wherein the hydrogen atom of             the secondary amino group in the pyrrolidine ring may be             replaced by             R²¹—C(═O)—P3₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—SIG-;         -   where L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid independently selected from Gly,             Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr,             Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or one             of the corresponding N-alkyl amino acids, preferably             N-methyl amino acids; in case there is more than one amino             acid P3, P3 may have the same or different amino acids, as             defined above;         -   where -SIG is a self-immolative group, which, upon cleavage             of the C(═O)—SIG bond provides the free secondary amine;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         C(═N—NH₂)—;     -   R³ represents -L-#1, -MOD or an optionally substituted alkyl,         cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl         group, preferably a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or C₆₋₁₀-aralkyl,         C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH((CH₂CH₂O)₁₋₂₀H) groups, 1-3 —NH₂ groups or 1-3         —(CH₂)₀₋₃Z groups,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where -MOD is represented as defined infra,         -   where n represents 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′ and         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NHC(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′, where         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH     -   R⁵ represents -L-#1, —H, -MOD, —NH₂, —NO₂, halogen (in         particular —F, —Cl, —Br), —CN, —CF₃, —OCF₃, —CH₂F, —CH₂F, —SH or         —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   R⁶ and R⁷ independently of one another represent —H, cyano,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy, —NO₂, —NH₂, —COOH or halogen (in particular —F, —Cl,         —Br),     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         C₄₋₁₀-cycloalkyl, fluoro-C₄₋₁₀-cycloalkyl, - or —(CH₂)₀₋₂—(HZ²),         -   where HZ² represents a 4- to 7-membered heterocycle having             up to two heteroatoms selected from the group consisting of             N, O and S, where each of these groups may be substituted by             OH, —COOH or —NH₂ or -L-#1;         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,     -   where one of the substituents R¹, R², R³, R⁴ R⁵, R⁸ and R¹⁰         represents (or in the case of R⁸ contains) -L-#1,     -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;     -   -MOD as defined supra represents (NR¹⁰)n-(G1)o-G2-G3,         -   where R¹⁰ represents —H or C₁-C₃-alkyl;         -   where G1 represents NH—C(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents NH—C(═O)— or

-   -   -    R¹⁰ does not represent —NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1;         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,             —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—,             —C(═O)—NR^(y)NR^(y)—, —C(═O)—, —CR^(x)═N—O—,             -   and where the hydrocarbon chain including any side                 chains may be substituted by             -   —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CNNH₂, sulphonamide,                 sulphone, sulphoxide or sulphonic acid,         -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,         -   where G3 represents —H or COOH, and         -   where -MOD preferably has at least one —COOH group;

    -   and the salts, solvates, salts of the solvates, and epimers         thereof.

-   -   -   where

    -   X₁ represents N, X₂ represents N and X₃ represents C; or

    -   X₁ represents N, X₂ represents C and X₃ represents N; or

    -   X₁ represents CH or CF, X₂ represents C and X₃ represents N; or

    -   X₁ represents NH, X₂ represents C and X₃ represents C; or

    -   X₁ represents CH, X₂ represents N and X₃ represents C

    -   R¹ represents —H, -MOD, -L-#1 or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂;

    -   R² represents -L-#1, H, -MOD, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′, and         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NHC(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;

    -   R⁴ represents -L-#1, —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂, and         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,

    -   or

    -   R⁴ represents a group of         -   R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or         -   R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)— or the             cathepsin cleavable group R²¹—(C═O)₍₀₋₁₎—(P3)₍₁₋₂₎-P2-,             -   where R²¹ represents H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or                 C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,                 C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,                 heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,                 C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,                 C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy                 group, which may be substituted one or more times with                 —NH₂,                 -   —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,                     N(alkyl)-C(═O)-alkyl, —SO₃H, —S(═O)₂NH₂,                     —S(═O)₂—N(alkyl)₂, —COOH, —C(═O)—NH₂,                     —C(═O)—N(alkyl)₂, or —OH, or represent —H or a group                     —(O)_(x)—(CH₂CH₂O)_(y)—R²²,             -   where x is 0 or 1 and             -   where v is a number from 1 to 20,             -   where R²² represents —H, -alkyl (preferably                 C₁₋₁₂-alkyl), —CH₂—COOH, —CH₂—CH₂—COOH, or                 —CH₂—CH₂—NH₂);             -   where P2 is an amino acid selected from Gly, Pro, Ala,                 Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys,                 Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His;             -   where P3 is an amino acid independently selected from                 Gly, Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp,                 Ser, Thr, Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline                 and His or one of the corresponding N-alkyl amino acids,                 preferably N-methyl amino acids; in case there is more                 than one amino acid P3, P3 may have the same or                 different amino acids, as defined above;         -   or

    -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, halogen (preferably —F or —Cl),             —NH₂, —SO₃H, —COOH, —SH, C₁₋₄-alkyl, C₁₋₄-haloalkyl,             C₁₋₄-alkoxy, hydroxy substituted C₁₋₄-alkyl,             —C(═O)—O—(C₁₋₄-alkyl) or —OH, and wherein the hydrogen atom             of the secondary amino group in the pyrrolidine ring may be             replaced by             R²¹—C(═O)—P3₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—SIG-;         -   where L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid independently selected from Gly,             Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr,             Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or one             of the corresponding N-alkyl amino acids, preferably             N-methyl amino acids;         -   where -SIG is a self-immolative group, which, upon cleavage             of the C(═O)—SIG bond provides the free secondary amine;

    -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         —C(═N—NH₂)—;

    -   R³ represents -L-#1, -MOD or an optionally substituted alkyl,         cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl         group, preferably -L-#1 or a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or         C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where -MOD is represented as defined infra,         -   where n represents 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′ and         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NHC(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′, where         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;

    -   R⁵ represents —H, —NH₂, —NO₂, halogen (in particular —F, —Cl,         —Br), —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;

    -   R⁶ and R⁷ independently of one another represent —H, cyano,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy or halogen (in particular —F, —Cl, —Br),

    -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₄₋₁₀-cycloalkyl,         fluoro-C₄₋₁₀-cycloalkyl, or optionally substituted oxetane; and

    -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;

    -   and the salts, solvates, salts of the solvates, and epimers         thereof.

By substitution of a hydrogen atom at R¹, R², R³, R⁴, R⁵ or R⁸ or at the pyrrolidine ring (R¹⁰) formed by R² and R⁴, in a manner known to the person of average skill the compound of the formula (IIa) or (II) in which none of the substituents R¹, R², R³, R⁴, R⁵, R⁸ and R¹⁰ represents -L-#1 may be attached to a linker. This gives conjugates of the formula (IIa) or (II) where one of the substituents R¹, R², R³, R⁴, R⁵, R⁸ or R¹⁰ represents -L-#1, L represents the linker and #1 represents the bond to the binder or the derivative thereof. If the KSP inhibitor according to formula (IIa) or (II) is conjugated with a binder, one of the substituents R¹, R², R³, R⁴, R⁵, R⁸ or R¹⁰ thus represents -L-#1, where L represents the linker and #1 represents the bond to the binder or the derivative thereof. That is, in the case of the conjugates one of the substituents R¹, R², R³, R⁴, R⁵, R⁸ and R¹⁰ represents -L-#1, where -L-#1 is attached to the binder, for example an antibody. With particular preference, one of the substituents R¹ and R³ represents -L-#1. In this embodiment, it is preferred that R⁴ represents —H, R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂COOH)—C(═O)— as defined above. In another preferred embodiment, substituent R⁴ represents -L-#1, wherein the Linker is cleavable at the nitrogen atom binding to R⁴ such that a primary amino group is generated by cleavage (corresponding to R⁴═—H). Corresponding cleavable groups are described below.

If R¹ is not —H, the carbon atom binding to R¹ is a stereocenter, which may be present in the L and/or D configuration, preferably in the L configuration.

If R² is not —H, the carbon atom binding to R² is a stereocenter, which may be present in the L and/or D configuration.

The binder is preferably a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or an antigen-binding fragment thereof or an anti-EGFR antibody or an antigen-binding fragment thereof. Particular preference is given to an anti-TWEAKR antibody which binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), in particular the anti-TWEAKR antibody TPP-2090, or the anti-Her2 antibody trastuzumab. All the antibodies described include aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

The compounds of the formula (IIa) or (II) in which one of the substituents R¹, R², R³, R⁴, R⁵ and R¹⁰ represents -L-#1 and in which

X¹ represents N, X₂ represents N and X₃ represents C;

X₁ represents CH or CF, X₂ represents C and X₃ represents N;

X₁ represents NH, X₂ represents C and X₃ represents C; or

X₁ represents CH, X₂ represents N and X₃ represents C

are particularly preferred,

in particular those in which

X₁ represents N, X₂ represents N and X₃ represents C; or X₁ represents CH, X₂ represents C and X₃ represents N. Particular preference is given to compounds in which X₁ represents CH, X₂ represents C and X₃ represents N.

For A, preference is given to —C(═O)—.

Preferred for R¹ are -L-#1, —H, —COOH, —C(═O)—NHNH₂, —(CH₂)₁₋₃NH₂, —C(═O)—NZ″(CH₂)₁₋₃NH₂ and —C(═O)—NZ″CH₂COOH, where Z″ represents —H or —NH₂.

R² and R⁴ represent —H, or R² represents —H and R⁴ represents R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— or R² and R⁴ together (with formation of a pyrrolidine ring) represent —CH₂—CHR¹⁰— or CHR¹⁰—CH₂—, where R¹⁰ represents —H or -L-#1.

Preferred for R³ is -L-#1 or C₁₋₁₀-alkyl-, which may optionally be substituted by —OH, —O-alkyl, —SH, —S-alkyl, —O—C(═O)-alkyl, —O—C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —NH—C(═O)—NH-alkyl, —S(O)_(n) alkyl —S(═O)₂—NH-alkyl, —NH-alkyl, —N(alkyl)₂ or —NH₂; where alkyl is preferably C₁₋₂-alkyl and n is 0, 1 or 2.

Preferred for R⁵ is -L-#1, —H or —F.

Preferred for R⁶ and R⁷, independently of one another, are —H, (optionally fluorinated) C₁₋₃-alkyl, (optionally fluorinated) C₂₋₄-alkenyl, (optionally fluorinated) C₂₋₄-alkynyl, hydroxy or halogen,

Preferred for R⁸ is a branched C₁₋₅-alkyl group, in particular a group of the formula —C(CH₃)₂—(CH₂)₀₋₂—R^(y), where R^(y) represents —H, —OH, —COOH, —NH₂ or -L-#1. Particular preference is given to the group of the formula —C(CH₃)₂—(CH₂)—R^(y), where R^(y) represents —H or -L-#1.

Preferred for R⁹ is —H or —F.

Particular preference is given to compounds of the formula (IIa) or (II) in which none or one of the substituents R¹, R², R³, R⁴, R⁵, R⁸ and R¹⁰ represents -L-#1, and

in which

X₁ represents N, X₂ represents N and X₃ represents C;

X₁ represents CH or CF, X₂ represents C and X₃ represents N;

X₁ represents NH, X₂ represents C and X₃ represents C; or

X₁ represents CH, X₂ represents N and X₃ represents C

A represents C(═O)—;

R¹ represents —H, —COOH, —C(═O)—NHNH₂, —(CH₂)₁₋₃NH₂, —C(═O)—NZ″(CH₂)₁₋₃—NH₂ and —C(═O)—NZ″CH₂COOH, where Z″ represents —H or —NH₂;

R² and R⁴ represent —H, or R² represents —H and R⁴ represents R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— (where P2 and P3 have the same meaning as defined above), or R² and R⁴ together (with formation of a pyrrolidine ring) represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—, where R¹⁰ represents H or -L-#1; R³ represents a phenyl group which may be mono- or polysubstituted by halogen (in particular F) or optionally fluorinated C₁₋₃-alkyl, or represents an optionally fluorinated C₁₋₁₀-alkyl group which may optionally be substituted by —OY⁴, —SY⁴, —O—C(═O)—Y⁴, —O—C(═O)—NH—Y⁴, —NH—C(═O)—Y⁴, —NH—C(═O)—NH—Y⁴, —S(═O)_(n)—Y⁴ (where n represents 0, 1 or 2), —S(═O)₂—NH—Y⁴, —NH—Y⁴ or —N(Y⁴)₂, where Y⁴ represents —H, phenyl (optionally mono- or polysubstituted by halogen (in particular F) or optionally fluorinated C₁₋₃-alkyl), or alkyl (where the alkyl group may be substituted by —OH, —COOH, and/or —NHC(═O)—C₁₋₃-alkyl and where alkyl preferably represents C₁₋₃-alkyl); where particularly preferably R³ may be substituted by —OH, —O-alkyl, —SH, —S-alkyl, —O—C(═O)-alkyl, —O—C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —NH—C(═O)—NH-alkyl, —S(═O)_(n)-alkyl (where n represents 0, 1 or 2), —S(═O)₂—NH-alkyl, —NH-alkyl, —N(alkyl)₂ or —NH₂ (where alkyl preferably means C₁₋₃-alkyl) R⁵ represents —H or —F; R⁶ and R⁷ independently of one another represent —H, (optionally fluorinated) C₁₋₃-alkyl, (optionally fluorinated) C₂₋₄-alkenyl, (optionally fluorinated) C₂₋₄-alkynyl, hydroxy or halogen; R⁸ represents a branched C₁₋₅-alkyl group; and R⁹ represents —H or —F.

Furthermore, it is preferred when (alone or in combination)

-   -   R¹ represents -L-#1, —COOH or —H,     -   R² and R⁴ independently of one another represent -L-#1 or —H; or         R² represents —H and R⁴ represents         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— (where P2         and P3 have the same meaning as defined above), or R² and R⁴         together (with formation of a pyrrolidine ring) represent         —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—, where R¹⁰ represents —H or -L-#1,     -   A represents —C(═O)—,     -   R³ represents —(CH₂)OH, —CH(CH₃)OH,         —CH₂—S—CH₂CH(COOH)NH—C(═O)—CH₃, —CH(CH₃)OCH₃, a phenyl group         which may be substituted by 1-3 halogen atoms, 1-3 amino groups         or 1-3 alkyl groups (which may optionally be halogenated), or         represents -L-#1,     -   R⁵ represents -L-#1 or —H,     -   R⁶ and R⁷ independently of one another represent —H, C₁₋₃-alkyl         or halogen; in particular, R⁶ and R⁷ represent —F;     -   R⁸ represents C₁₋₄-alkyl (preferably tert-butyl); and/or     -   R⁹ represents —H,     -   where one of the substituents R¹, R², R³, R⁴, R⁵ and R¹⁰         represents -L-#1.

Additionally, in accordance with the invention it is preferred when

-   -   R¹ represents -L-#1, —COOH or —H,     -   R² and R⁴ independently of one another represent -L-#1 or —H, or         R² represents —H and R⁴ represents         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— (where P2         and P3 have the same meaning as defined above), or R² and R⁴         together (with formation of a pyrrolidine ring) represent         —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—, where R¹⁰ represents —H or -L-#1,     -   A represents —C(═O)—,     -   R³ represents —(CH₂)OH, —CH(CH₃)OH,         —CH₂—S—CH₂CH(COOH)NH—C(═O)—CH₃, —CH(CH₃)OCH₃, a phenyl group         which may be substituted by 1-3 halogen atoms, 1-3 amino groups         or 1-3 alkyl groups (which may optionally be halogenated), or         represents -L-#1,     -   R⁵ represents -L-#1 or —H,     -   R⁶ and R⁷ independently of one another represent —H, C₁₋₃-alkyl         or halogen; in particular, R⁶ and R⁷ represent —F;     -   R⁸ represents C₁₋₄-alkyl (preferably tert-butyl); and     -   R⁹ represents —H,     -   where one of the substituents R¹, R², R³, R⁴, R⁵ and R¹⁰         represents -L-#1.

Other particularly preferred compounds have the formula (IIIa) or (III) below:

-   -   where     -   X₁ represents N, X₂ represents N and X₃ represents C; or     -   X₁ represents N, X₂ represents C and X₃ represents N; or     -   X₁ represents CH or CF, X₂ represents C and X₃ represents N; or     -   X₁ represents NH, X₂ represents C and X₃ represents C; or     -   X₁ represents CH, X₂ represents N and X₃ represents C     -   (with X₁ representing CH, X₂ representing C and X₃ representing         N being preferred);     -   R¹ represents —H, -L-BINDER, -MOD or —(CH₂)₀₋₃Z,         -   where Z represents —H, —NHY³, —OY³, —SY³, halogen,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′, and         -   where Y³ represents H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—(C═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R² represents -L-BINDER, —H, -MOD, —C(═O)—CHY⁴—NHY⁵ or         —(CH₂)₀₋₃Z,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R⁴ represents -L-BINDER, —H, —C(O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R⁴ represents a group of         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—,         -   where R²¹ represents —H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group,             which may be substituted one or more times with —NH₂,             —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,             —N(alkyl)-C(═O)-alkyl, —SO₃H, —SO₂NH₂, —S(═O)₂—N(alkyl)₂,             —COOH, —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or —OH, or is —H or a             group —O_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1,         -   where v is a number from 1 to 20, and         -   where R²² represents —H, -alkyl (preferably C1-12-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂;         -   where P2 is an amino acid selected from Gly, Pro, Ala, Val,             Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,             Asp, Glu, Lys, Arg, citrulline and His;         -   where P3 is an amino acid independently selected from Gly,             Pro, Ala, Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr,             Cys, Asn, Gln, Asp, Glu, Lys, Arg, citrulline and His or one             of the corresponding N-alkyl amino acids, preferably             N-methyl amino acids;     -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents -L-BINDER, —H, —NH₂, —SO₃H, —COOH, —SH             or —OH;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         —C(═N—NH₂)—;     -   R³ represents -L-BINDER, -MOD or an optionally substituted         alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl,         heterocycloalkyl group, preferably -L-BINDER, or a C₁₋₁₀-alkyl,         C₆₋₁₀-aryl or C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl,         C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or C₅₋₁₀-heterocycloalkyl group which         may be substituted by 1-3 —OH groups, 1-3 halogen atoms, 1-3         halogenated alkyl groups (each having 1-3 halogen atoms), 1-3         —O-alkyl groups, 1-3 —SH groups, 1-3 —S-alkyl groups, 1-3         —O—C(═O)-alkyl groups, 1-3 —O—C(═O)—NH-alkyl groups, 1-3         —NH—C(═O)-alkyl groups, 1-3 —NH—C(═O)—NH-alkyl groups, 1-3         —S(═O)_(n)-alkyl groups, 1-3 —S(═O)₂—NH-alkyl groups, 1-3         —NH-alkyl groups, 1-3 —N(alkyl)₂ groups, 1-3 —NH₂ groups or 1-3         —(CH₂)₀₋₃Z groups,         -   where n is 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH         -   where “alkyl” preferably represents C₁₋₁₀-alkyl);     -   R⁵ represents -L-BINDER, —H, —NH₂, —NO₂, halogen (in particular         F, Cl, Br), —CN, —CF₃, —OCF₃, —CH₂F, —CH₂F, —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   R⁶ and R⁷ independently of one another represent —H, cyano,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy, —NO₂, —NH₂, —COOH or halogen (in particular —F, —Cl,         —Br),     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         C₄₋₁₀-cycloalkyl, fluoro-C₄₋₁₀-cycloalkyl or —(CH₂)₀₋₂—(HZ²),         -   where HZ² represents a 4- to 7-membered heterocycle having             up to two heteroatoms selected from the group consisting of             N, O and S (preferably oxetane), where each of these groups             may be substituted by —OH, —COOH or —NH₂ or -L-BINDER;     -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;     -   L represents a linker,     -   BINDER represents a binder or a derivative thereof, where the         binder may optionally be attached to a plurality of active         compound molecules,         -   where one representative of R¹, R², R³ R⁴, R⁵ and R⁸             represents -L-binder;     -   -MOD represents —(NR¹⁰)_(n)-(G1)_(o)-G2-H, where         -   where R¹⁰ represents —H or C₁-C₃-alkyl;         -   where G1 represents —NHC(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents —NH—C(═O)— or

-   -   -    R¹⁰ does not represent NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1; and         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,             —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—,             —C(═O)—NR^(y)NR^(y)—,         -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid, or             represents —C(═O)—, —CR^(x)═N—O—         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,         -   where the hydrocarbon chain including any side chains may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   and where -MOD preferably has at least one group —COOH;

    -   and the salts, solvates and salts of the solvates thereof.

In the case of binder conjugates of the KSP inhibitors of the formula (IIIa), at most one representative of R¹, R², R³ R⁴, R⁵, R⁸ and R¹⁰ (alternatively to one of the conditions given above) may represent -L-BINDER, where L represents a linker and BINDER represents a binder or a derivative thereof, where the binder may optionally be attached to a plurality of active compound molecules.

-   -   where     -   X₁ represent N, X₂ represents N and X₃ represents C, or     -   X₁ represents CH, X₂ represents C and X₃ represents N or     -   X₁ represents NH, X₂ represents C and X₃ represents C, or     -   X₁ represents CH, X₂ represents N and X₃ represents C;     -   R¹ represents —H, -L-BINDER or —(CH₂)₀₋₃Z,         -   where Z represents —H, —NHY³, —OY³, —SY³, halogen,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′, and         -   where Y³ represents H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—(C═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R² and R⁴ independently of one another represent -L-BINDER, —H,         —C(O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents -L-#1, —H, —NH₂, —SO₃H, —COOH, —SH, or             —OH;         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         —C(═N—NH₂)—;     -   R³ represents -L-BINDER or an optionally substituted alkyl,         cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl         group, preferably L-BINDER, or a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or         C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where n is 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH         -   where “alkyl” preferably represents C₁₋₁₀-alkyl);     -   R⁵ represents -L-BINDER, —H, —NH₂, —NO₂, halogen (in particular         F, Cl, Br), —CN, —CF₃, —OCF₃, —CH₂F, —CH₂F, —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   R⁶ and R⁷ independently of one another represent —H, cyano,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy, or halogen (in particular —F, —Cl, —Br),     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₄₋₁₀-cycloalkyl,         fluoro-C₄₋₁₀-cycloalkyl or optionally substituted oxetane;     -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;     -   -L represents a linker and     -   -BINDER represents a binder or a derivative thereof, where the         binder may optionally be attached to a plurality of active         compound molecules,         -   where one representative of R¹, R², R³ R⁴, R⁵, R⁸ and R¹⁰             represents -L-binder;             and the salts, solvates and salts of the solvates thereof.

Furthermore, preference according to the invention is given to conjugate of the following KSP inhibitors:

where X₁, X₂, X₃ have the same meaning as in formula (IIIa) or (III) (where preferably X₁ represents —CH—, X₂ represents —C— and X₃ represent —N—), R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ have the same meaning as in formula (IIIa) or (III), A represents C(═O)—, B represents a single bond, —O—CH₂ or —CH₂—O— and R²⁰ represents —NH₂, —F, —CF₃ or —CH₃ and n represents 0, 1 or 2.

where X₁, X₂, X₃ have the same meaning as in formula (IIIa) or (III) (where preferably X₁ represents —CH—, X₂ represents —C— and X₃ represents —N—); A, R¹, R³, R⁶, R⁷, R⁸ and R⁹ have the same meaning as in formula (IIIa) or (III), A preferably represents —C(═O)— and R³ represents CH₂OH, —CH₂OCH₃, —CH(CH₃)OH or —CH(CH₃)OCH₃.

where X₁, X₂, X₃ have the same meaning as in formula (IIIa) or (III) (where preferably X₁ represents —CH—, X₂ represents —C— and X₃ represents —N—); A, R³, R⁶, R⁷, R⁸ and R⁹ have the same meaning as in formula (IIIa) or (III), where A preferably represents —C(═O)— and R³ represents —CH₂—S_(x)(CH₂)₀₋₄—CHY⁵—COOH, where x is 0 or 1 and Y⁵ represents —H or —NHY⁶, where Y⁶ represents —H or C(═O)—CH₃.

where X₁ represents —CH—, X₂ represents —C— and X₃ represents —N—; A, R³, R⁴, R⁶, R⁷, R⁸ and R⁹ have the same meaning as in formula (IIIa) or (III) and R¹ represents -L-BINDER.

Furthermore, it is preferred when in the compounds of the formulae (III), (IIIa), (IIIb), (IIIc), (IIId) and (IIIe) (alone or in combination):

-   -   Z represents Cl or Br;     -   R¹ represents —(CH₂)₀₋₃Z, where Z represents —C(═O)—NY¹Y², where         Y² represents —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ and Y¹ represents —H,         —NH₂ or —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′;     -   Y¹ represents —H, Y² represents —(CH₂CH₂O)₃—CH₂CH₂Z′ and Z′         represents —COOH;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′ and Z′ represents         —(C(═O)—NHCHY⁴)₂COOH;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′, Z′ represents         —(C(═O)—NHCHY⁴)₂COOH and one of the Y⁴ radicals represents         i-propyl and the other —(CH₂)₃—NH—C(═O)—NH₂;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′, Z′ represents         —(C(═O)—NHCHY⁴)₂COOH and one of the Y⁴ radicals represents —CH₃         and the other —(CH₂)₃—NH—C(═O)—NH₂;     -   Y⁴ represents linear or branched C₁₋₆-alkyl which is optionally         substituted by —NH—C(═O)—NH₂;     -   at least one Y⁴ representative is selected from the group         consisting of i-propyl and —CH₃;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′, Z′ represents         —C(═O)—NHCHY⁴COOH and Y⁴ represents aryl or benzyl which are         optionally substituted by —NH₂;     -   Y⁴ represents aminobenzyl;     -   R² represents —(CH₂)₀₋₃Z and Z represents —SY³;     -   R⁴ represents —C(═O)—CHY⁴—NHY⁵ and Y⁵ represents —H;     -   R⁴ represents —C(═O)—CHY⁴—NHY⁵ and Y⁵ represents         —C(═O)—CHY⁶—NH₂;     -   R⁴ represents R²¹-L-Ala-L-Ala-L-Asn-#;     -   Y⁴ represents linear or branched C₁₋₆-alkyl which is optionally         substituted by —NH—C(═O)—NH₂.

Furthermore, it is preferred when R¹, R² or R³ in formula (IIa) or (IIIa) represents -MOD, in particular when R⁴ represents -L-#1 or -L-BINDER (in particular when -L is a cleavable linker which cleaves directly at —N—R⁴ or —N-L-#1 or -L-BINDER, such that R⁴ or L is replaced by —H).

Particularly preferably, R³ represents -MOD and R¹ or R⁴ represents -L-#1 or -L-BINDER,

-   -   where -MOD represents (NR¹⁰)_(n)-(G1)_(o)-G2-H,         -   where R¹⁰ represents —H or C₁-C₃-alkyl;         -   where G1 represents —NH—C(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents —NH—C(═O)— or

-   -   -    R¹⁰ does not represent NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1; and         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,             —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—,             —C(═O)—NR^(y)NR^(y)—,         -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid, or             represents —C(═O)—, —CR^(x)═N—O—         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,         -   where the hydrocarbon chain including any side chains may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid, and         -   where -MOD preferably has at least one group COOH.

Particularly preferably, the group -MOD has a (preferably terminal) —COOH group, for example in a betaine group. Preferably, the group -MOD has the formula —CH₂—S_(x)—(CH₂)₀₋₄—CHY⁵—COOH where x is 0 or 1, and Y⁵ represents —H or —NHY⁶, where Y⁶ represents —H or C(═O)—CH₃.

Other particularly preferred compounds have the formula (IV) below:

where

-   -   X₁ represents N, X₂ represents C and X₃ represents N;     -   R¹ represents —H, -L-BINDER or —(CH₂)₀₋₃Z,         -   where Z represents —H, —NHY³, —OY³, —SY³, halogen,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ or —CH(CH₂W)Z′,         -   where Y³ represents H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—(C═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R² and R⁴ independently of one another represents -L-BINDER, —H,         —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,     -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents L-#1, —H, —NH₂, —SO₃H, —COOH, —SH, or             —OH;         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         C(═N—NH₂)—;     -   R³ represents -L-BINDER or an optionally substituted alkyl,         cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl         group, or CH₂—S_(x)—(CH₂)₀₋₄—CHY⁵—COOH,         -   where x is 0 or 1,         -   where Y⁵ represents —H or —NHY⁶,         -   where Y⁶ represents —H or —C(═O)—CH₃,         -   preferably -L-BINDER, or a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or             C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl             or C₅₋₁₀-heterocycloalkyl group which may be substituted by             1-3 —OH groups, 1-3 halogen atoms, 1-3 halogenated alkyl             groups (each having 1-3 halogen atoms), 1-3 —O-alkyl groups,             1-3 —SH groups, 1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl             groups, 1-3 —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl             groups, 1-3         -   —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3             —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3             —N(alkyl)₂ groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where n is 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH         -   where “alkyl” preferably represents C₁₋₁₀-alkyl);     -   R⁵ represents -L-BINDER, —H, —NH₂, —NO₂, halogen (in particular         F, Cl, Br), —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where L represents a linker,         -   BINDER represents a binder or a derivative thereof, where             the binder may optionally be attached to a plurality of             active compound molecules;     -   R⁶ and R⁷ independently of one another represent —H, cyano,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy or halogen (in particular —F, —Cl, —Br);     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₄₋₁₀-cycloalkyl,         fluoro-C₄₋₁₀-cycloalkyl or optionally substituted oxetane;     -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;     -   and the salts, solvates, salts of the solvates, and epimers         thereof;     -   with the proviso that R¹, R² and R⁴ do not simultaneously         represent —H.

Furthermore, it is preferred when in the formula (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IV) (alone or in combination):

-   -   Z represents Cl or Br;     -   R¹ represents —(CH₂)₀₋₃Z, where Z represents —C(═O)—NY¹Y², where         Y² represents —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ and Y¹ represents H, NH₂         or —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′;     -   Y¹ represents —H, Y² represents —(CH₂CH₂O)₃—CH₂CH₂Z′ and Z′         represents —COOH;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′ and Z′ represents         —(C(═O)—NHCHY⁴)₂COOH;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′, Z′ represents         —(C(═O)—NHCHY⁴)₂COOH and one Y⁴ representative represents         i-propyl and the other represents —(CH₂)₃—NH—C(═O)—NH₂;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′, Z′ represents         —(C(═O)—NHCHY⁴)₂COOH and one Y⁴ representative represents —CH₃         and the other represents —(CH₂)₃—NH—C(═O)—NH₂;     -   Y⁴ represents linear or branched C₁₋₆-alkyl which is optionally         substituted by —NH—C(═O)—NH₂;     -   at least one Y⁴ representative is selected from the group         consisting of i-propyl and —CH₃;     -   Y¹ represents —H, Y² represents —CH₂CH₂Z′, Z′ represents         —C(═O)—NHCHY⁴COOH and Y⁴ represents aryl or benzyl which are         optionally substituted by —NH₂;     -   Y⁴ represents aminobenzyl;     -   R² represents —(CH₂)₀₋₃Z and Z represents —SY³;     -   R⁴ represents —C(═O)—CHY⁴—NHY⁵ and Y⁵ represents —H;     -   R⁴ represents —C(═O)—CHY⁴—NHY⁵ and Y⁵ represents         —C(═O)—CHY⁶—NH₂;     -   R⁴ represents R²¹-L-Ala-L-Ala-L-Asn-#;     -   Y⁴ represents linear or branched C₁₋₆-alkyl which is optionally         substituted by —NH—C(═O)—NH₂.

Preference is furthermore given to compounds of the formula (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IV)

-   -   where     -   X₁ represents N, X₂ represents N and X₃ represents C; or     -   X₁ represents N, X₂ represents C and X₃ represents N; or     -   X₁ represents CH or CF, X₂ represents C and X₃ represents N; or     -   X₁ represents NH, X₂ represents C and X₃ represents C; or     -   X₁ represents CH or CF, X₂ represents N and X₃ represents C;     -   (with X₁ representing CH, X₂ representing C and X₃ representing         N being preferred);     -   R¹ represents H, -L-#1 or -L-BINDER, -MOD or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or             —CH(CH₂W)Z′,         -   where Y³ represents H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—(C═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R² represents —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R⁴ represents —H or R²¹-L-Ala-L-Ala-L-Asn-;         -   where R²¹ represents —H, C₅₋₁₀-aryl- or C₆₋₁₀-aralkyl-,             C₅₋₁₀-heteroalkyl-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-,             C₅₋₁₀-heterocycloalkyl-, heteroaryl-, heteroaryl-alkyl-,             C₁₋₁₀-alkoxy-, C₆₋₁₀-aryloxy-oder C₆₋₁₀-aralkoxy-,             C₅₋₁₀-heteroalkoxy-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-,             C₅₋₁₀-heterocycloalkoxy group, which may be substituted one             or more times with —NH₂, —NH-alkyl, —N(alkyl)₂,             —NH—C(═O)-alkyl, —N(alkyl)-C(═O)-alkyl, —SO₃H, —SO₂NH₂,             —S(═O)₂—N(alkyl)₂, —COOH, —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or             —OH, or is —H or a group —O_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1,         -   where v is a number from 1 to 20, and         -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         —C(═N—NH₂)—;     -   R³ represents -L-#1 or -L-BINDER, -MOD or an optionally         substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl,         heterocycloalkyl group, preferably -L-BINDER, or a C₁₋₁₀-alkyl,         C₆₋₁₀-aryl or C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl,         C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or C₅₋₁₀-heterocycloalkyl group which         may be substituted by 1-3 —OH groups, 1-3 halogen atoms, 1-3         halogenated alkyl groups (each having 1-3 halogen atoms), 1-3         —O-alkyl groups, 1-3 —SH groups, 1-3 —S-alkyl groups, 1-3         —O—C(═O)-alkyl groups, 1-3 —O—C(═O)—NH-alkyl groups, 1-3         —NH—C(═O)-alkyl groups, 1-3 —NH—C(═O)—NH-alkyl groups, 1-3         —S(═O)_(n)-alkyl groups, 1-3 —S(═O)₂—NH-alkyl groups, 1-3         —NH-alkyl groups, 1-3 —N(alkyl)₂ groups, 1-3 —NH₂ groups or 1-3         —(CH₂)₀₋₃Z groups,         -   where n is 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH         -   where “alkyl” preferably represents C₁₋₁₀-alkyl);     -   R⁵ represents —H, -MOD, —NH₂, —NO₂, halogen (in particular F,         Cl, Br), —CN, —CF₃, —OCF₃, —CH₂F, —CH₂F, —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   R⁶ and R⁷ independently of one another represent —H, cyano,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy, —NO₂, —NH₂, —COOH or halogen (in particular —F, —Cl,         —Br),     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         C₄₋₁₀-cycloalkyl or fluoro-C₄₋₁₀-cycloalkyl,         -   where one of the substituents R¹ and R³ represents -L-#1 or             -L-BINDER,     -   L represents the linker,     -   #1 represents the bond to the binder or derivative thereof,     -   BINDER represents the binder,     -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;     -   -MOD represents —(NR¹⁰)n-(G1)o-G2-G3,         -   where R¹⁰ represents —H or C₁-C₃-alkyl;         -   where G1 represents —NH—C(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents —NH—C(═O)— or

-   -   -    R¹⁰ does not represent —NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1; and         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,             —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—,             —C(═O)—NR^(y)NR^(y)—,         -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid, or             represents —C(═O)—, —CR^(x)═N—O—         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,         -   where the hydrocarbon chain including any side chains may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   where G3 represents —H or —COOH, and         -   where -MOD preferably has at least one group —COOH;

    -   and the salts, solvates, salts of the solvates, and epimers         thereof.

Preference is furthermore given to compounds of the formula (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or (IV) in which

-   -   X₁ represents N, X₂ represents N and X₃ represents C; or     -   X₁ represents N, X₂ represents C and X₃ represents N; or     -   X₁ represents CH or CF, X₂ represents C and X₃ represents N; or     -   X₁ represents NH, X₂ represents C and X₃ represents C; or     -   X₁ represents CH or CF, X₂ represents N and X₃ represents C;     -   (with X₁ representing CH, X₂ representing C and X₃ representing         N being preferred);     -   R¹ represents —H, -L-#1 or -L-BINDER, -MOD or —(CH₂)₀₋₃Z,         -   where Z represents —H, —NHY³, —OY³, —SY³, halogen,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′, and         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—(C═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R² represents —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R⁴ represents —H or R²¹-L-Ala-L-Ala-L-Asn-;         -   where R²¹ represents —H, C₅₋₁₀-aryl- or C₆₋₁₀-aralkyl-,             C₅₋₁₀-heteroalkyl-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-,             C₅₋₁₀-heterocycloalkyl-, heteroaryl-, heteroaryl-alkyl-,             C₁₋₁₀-alkoxy-, C₆₋₁₀-aryloxy-oder C₆₋₁₀-aralkoxy-,             C₅₋₁₀-heteroalkoxy-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-,             C₅₋₁₀-heterocycloalkoxy group, which may be substituted one             or more times with —NH₂, —NH-alkyl, —N(alkyl)₂,             —NH—C(═O)-alkyl, —N(alkyl)-C(═O)-alkyl, —SO₃H, —SO₂NH₂,             —S(═O)₂—N(alkyl)₂, —COOH, —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or             —OH, or is —H or a group —O_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1,         -   where v is a number from 1 to 20, and         -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         C(═N—NH₂)—;     -   R³ represents -L-#1 or -L-BINDER, -MOD or an optionally         substituted alkyl, cycloalkyl, aryl, heteroaryl, heteroalkyl,         heterocycloalkyl group, preferably C₁₋₁₀-alkyl, C₆₋₁₀-aryl or         C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where n is 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH         -   where “alkyl” preferably represents C₁₋₁₀-alkyl);     -   R⁵ represents -MOD, —H, —NH₂, —NO₂, halogen (in particular F,         Cl, Br), —CN, —CF₃, —OCF₃, —CH₂F, —CH₂F, —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   R⁶ and R⁷ independently of one another represent —H or halogen         (in particular —F, —Cl, —Br),     -   R⁸ represents C₁₋₁₀-alkyl or fluoro-C₁₋₁₀-alkyl,     -   where one of the substituents R¹ and R³ represents -L-#1 or         -L-BINDER,     -   L represents the linker,     -   #1 represents the bond to the binder or derivative thereof,     -   BINDER represents the binder,     -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;     -   -MOD represents CH₂—S_(x)—(CH₂)₀₋₄—CHY⁵—COOH,         -   where x is 0 or 1,         -   where Y⁵ represents H or NHY⁶,         -   where Y⁶ represents H or —C(═O)—CH₃,     -   and the salts, solvates, salts of the solvates, and epimers         thereof.

Particular preference according to the invention is given to the conjugate of the following compounds of the formulae V, VI and VII, where R¹, R², R³, R⁴ and R⁵ have the meanings mentioned above (as mentioned, for example for formula (IIa) or (IIIa)):

Particular preference is given to the compounds of the formulae V, VI, VII where R¹ and R⁵ represent —H or -L-#1; R² and R⁴ independently of one another represent -L-#1 or —H or R² and R⁴ together (with formation of a pyrrolidine ring) represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—, R¹⁰ represents —H or -L-#1; and R³ represents —CH₂OH, —CH(CH₃)OH or -L-#1, where one of the substituents R¹, R², R³, R⁴, R⁵ and R¹⁰ represents -L-#1. Especially preferred are the corresponding compounds of the formula VI.

Preferred antibody drug conjugates of the present invention (ADCs) are those of the following Formula VIII:

wherein

-   m is a number from 0 to 2st; -   n is 0 or 1; -   X represents C(═O)—NH₂ or COOH; -   L_(a) represents a self-immolative linker; -   L_(c) represents a linker. -   A₁ is a residue derived from one of the amino acids Gly, Pro, Ala,     Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,     Asp, Glu, Lys, Arg, Citrulline and His; -   A₂ is a residue derived from one of the amino acids Gly, Pro, Ala,     Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,     Asp, Glu, Lys, Arg, Citrulline and His or one of the corresponding     N-alkyl-amino acids, preferably N-methyl-amino acid (when there is     more than one P3, P3 can have different meanings) -   D1 is KSP according to Formula II or IIa (ie. without linker L); -   R represents Z₁—(C═O)q-,     -   wherein q is 0 or 1 and -   Z₁ represents a C₁₋₁₀-alkyl-, C₅₋₁₀-Aryl- or C₆₋₁₀-aralkyl-,     C₅₋₁₀-heteroalkyl-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-,     C₅₋₁₀-heterocycloalkyl-, heteroaryl-, heteroaryl-alkyl-,     C₅₋₁₀-heteroaryl-alkoxy-, C₁₋₁₀-alkoxy-, C₆₋₁₀-aryloxy- or     C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-,     C₅₋₁₀-heterocycloalkoxy group, which may be substituted one or more     times with —NH₂, —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,     —N(alkyl)-C(═O)-alkyl, —SO₃H, —S(═O)₂—NH₂, —S(═O)₂—N(alkyl)₂, —COOH,     —C(═O)—NH₂, —C(═O)—N(alkyl)₂, —OH, —H or a group     —O_(x)—(CH₂CH₂O)_(y)—R¹,     -   wherein x is 0 or 1,     -   wherein v is a number from 1 to 20,     -   wherein R¹ represents —H, -alkyl (preferably C₁₋₁₂-alkyl),         —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂, -   AB represents an antibody, -   s is a number from 1 to 20, preferably 2 to 8, particularly     preferred 2 to 4 as e.g. 4.

Preferred antibody prodrug conjugates of the present invention (APDCs) are those of the following Formula IX:

-   wherein -   m is 0, 1 or 2; -   n is 0 or 1; -   X represents —C(═O)—NH₂ or —COOH; -   L_(a) represents a self-immolative linker; -   L_(b) represents a linker. -   A₁ is a residue derived from one of the amino acids Gly, Pro, Ala,     Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,     Asp, Glu, Lys, Arg, Citrulline and His; -   A₂ is a residue derived from one of the amino acids Gly, Pro, Ala,     Val, Nva, Leu, Ile, Met, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln,     Asp, Glu, Lys, Arg, Citrulline and His or one of the corresponding     N-alkyl-amino acids, preferably N-methyl-amino acid (when there is     more than one P3, P3 can have different meanings) -   D₁ is KSP according to Formula II or IIa (ie. without linker L); -   R represents Z₁—(C═O)q-,     -   wherein q is 0 or 1 and -   Z₁ represents a C₁₋₁₀-alkyl-, C₅₋₁₀-Aryl- or C₆₋₁₀-aralkyl-,     C₅₋₁₀-heteroalkyl-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-,     C₅₋₁₀-heterocycloalkyl-, heteroaryl-, heteroaryl-alkyl-,     C₅₋₁₀-heteroaryl-alkoxy-, C₁₋₁₀-alkoxy-, C₆₋₁₀-aryloxy- or     C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-,     C₅₋₁₀-heterocycloalkoxy group, which may be substituted one or more     times with —NH₂, —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,     —N(alkyl)-C(═O)-alkyl, —SO₃H, —S(═O)₂—NH₂, —S(═O)₂—N(alkyl)₂, —COOH,     —C(═O)—NH₂, —C(═O)—N(alkyl)₂, or —OH, —H     -   or a group —O_(x)—(CH₂CH₂O)_(y)—R¹,     -   wherein x is 0 or 1,     -   wherein v is a number from 1 to 20,     -   wherein R¹ represents —H, -alkyl (preferably C₁₋₁₂-alkyl),         —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂, -   AB represents an antibody, -   s is a number from 1 to 20, preferably 2 to 8, particularly     preferred 2 to 4 as e.g. 4.     Linkers

The literature discloses various options for covalently coupling (conjugating) organic molecules to binders in a site specific homogenous manner such as, for example antibodies (see, for example, (Sochaj et al., Biotechnology Advances, Article in press (2015), Panowski et al., MAbs 6, 34-45 (2014))

Preference according to the invention is given to conjugation of the KSP inhibitors to an antibody via acceptor glutamine residues of the antibody using transglutaminase. Such acceptor glutamines can be introduced by engineering of the antibody by mutations or by generation of aglycosylated antibodies. The number of the said acceptor glutamine residue in the antibody is preferably 2 or 4. For coupling, use is made of linkers. Linkers can be categorized into the group of the linkers which can be cleaved in vivo and the group of the linkers which are stable in vivo (see L. Ducry and B. Stump, Bioconjugate Chem. 21, 5-13 (2010)). The linkers which can be cleaved in vivo have a group which can be cleaved in vivo, where, in turn, a distinction may be made between groups which are chemically cleavable in vivo and groups which are enzymatically cleavable in vivo. “Chemically cleavable in vivo” and “enzymatically cleavable in vivo” means that the linkers or groups are stable in circulation and are cleaved only at or in the target cell by the chemically or enzymatically different environment therein (lower pH; elevated glutathione concentration; presence of lysosomal enzymes such as legumain, cathepsin or plasmin, or glyosidases such as, for example, ß-glucuronidases), thus releasing the low-molecular weight KSP inhibitor or a derivative thereof. Groups which can be cleaved chemically in vivo are in particular disulphide, hydrazone, acetal and aminal; groups which can be cleaved enzymatically in vivo are in particular the 2-8-oligopeptide group, especially a dipeptide group or glycoside. Peptide cleavage sites are disclosed in Bioconjugate Chem. 2002, 13, 855-869, and Bioorganic & Medicinal Chemistry Letters 8 (1998) 3341-3346 and also Bioconjugate Chem. 1998, 9, 618-626. These include, for example, valine-alanine, valine-lysine, valine-citrulline, alanine-lysine and phenylalanine-lysine (optionally with additional amide group).

Linkers which are stable in vivo are distinguished by a high stability (less than 5% metabolites after 24 hours in plasma) and do not have the chemically or enzymatically in vivo cleavable groups mentioned above.

The linker -L- preferably has one of the basic structures (i) to (iv) below:

-   -   (i) (C═O)_(m)-SG1-L1-L2-     -   (ii) (C═O)_(m)-L1-SG-L1-L2-     -   (iii) (C═O)_(m)-L1-L2-     -   (iv) (C═O)_(m)-L1-SG-L2         where m is 0 or 1; SG is a (chemically or enzymatically) in vivo         cleavable group (in particular disulphide, hydrazone, acetal and         aminal; or a 2-8-oligopeptide group which can be cleaved by         cathepsin or plasmin), SG1 is an oligopeptide group or         preferably a dipeptide group, L1 independently of one another         represent in vivo stable organic groups, and L2 represents a         coupling group to the binder.

Particular preference according to the invention is given to the basic linker structure (iii), in particular when the binder is an antibody. Via metabolization, the administration of a conjugate according to the invention having a basic linker structure (iii) and coupling of the linker to a glutamine residue of the binder protein or peptide using transglutaminase leads to glutamine derivatives of the formulae below:

where L1 is in each case attached to the low-molecular weight KSP inhibitor, for example a compound of the formula (I), (IIa), (II), (III), (IIIa), (IIIb), (IIIc), (IIId), (IIIe) or any of (IV) to (IX).

According to the invention, L1 is preferably represented by the formula ^(#1)—(NR¹⁰)_(n)-(G1)_(o)-G3^(#2) where R¹⁰ represents —H, —NH₂ or C₁-C₃-alkyl; G1 represents NH—C(═O)—, —C—(═O)—NH— or

(R¹⁰ is preferably not —NH₂, if G1 represents NH—C(═O)— or

n is 0 or 1; o is 0 or 1; and

G3 represents a bond or an optionally substituted linear or branched hydrocarbon chain which has 1 to 100 carbon atoms from arylene groups and/or linear and/or branched and/or cyclic alkylene groups and which may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)—C(═O)—, —C(NH)NR^(y)—, —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—, —C(═O)—NR^(y)NR^(y)— (where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid), —C(═O)—, —CR^(x)═N—O— (where R^(x) represents —H, C₁-C₃-alkyl or phenyl) and/or a 3- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from the group consisting of N, O and S, —S(═O)— or S(═O)₂— (preferably

where the hydrocarbon chain including any side chains may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

G3 represents a bond or an optionally substituted linear or branched hydrocarbon chain having 1 to 100 carbon atoms from arylene groups and/or linear and/or branched and/or cyclic alkylene groups and which may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NH—, —C(═O)—, —NH—C(═O)—, —C(═O)—NH—, —NMe-, —NHNH—, —S(═O)₂—NHNH—, —C(═O)—NHNH— and a 5- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from the group consisting of N, O and S, or —S(═O)— (preferably

where the side chains, if present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

G3 represents a bond or an optionally substituted a linear or branched hydrocarbon chain having 1 to 100 carbon atoms from arylene groups and/or linear and/or branched and/or cyclic alkylene groups and which may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NH—, —C(═O)—, —NH—C(═O)—, —C(═O)—NH—, —NMe-, —NHNH—, —S(═O)₂—NHNH—, —C(═O)—NHNH—, —CR^(x)═N—O— (where R^(x) represents H, C₁-C₃-alkyl or phenyl) and a 3- to 10-membered, for example 5- to 10-membered, aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from the group consisting of N, O and S, —S(═O)— or S(═O)₂— (preferably

where the hydrocarbon chain including the side chains, if present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

Further interrupting groups in G3 are preferably

where Rx represents H, C₁-C₃-alkyl or phenyl.

Here, #¹ is the bond to the KSP inhibitor and #² is the bond to the coupling group to the binder (e.g. L2).

A linearlinear or branched hydrocarbon chain of arylen groups and/or linearlinear and/or branched and/or cyclic alkylene groups generally comprises a α,ω-divalent alkyl radical having the respective number of carbon atoms stated. Examples which may be mentioned as being preferred are: methylene, ethane-1,2-diyl (1,2-ethylene), propane-1,3-diyl (1,3-propylene), butane-1,4-diyl (1,4-butylene), pentane-1,5-diyl (1,5-pentylene), hexane-1,6-diyl (1,6-hexylene), heptane-1,7-diyl (1,7-hexylene), octane-1,8-diyl (1,8-octylene), nonane-1,9-diyl (1,9-nonylene), decane-1,10-diyl (1,10-decylene). However, the alkylene groups in the hydrocarbon chain may also be branched, i.e. one or more hydrogen atoms of the linear alkylene groups mentioned above may optionally be substituted by C₁₋₁₀-alkyl groups, thus forming side chains. The hydrocarbon chain may furthermore contain cyclic alkylene groups (cycloalkanediyl), for example 1,4-cyclohexanediyl or 1,3-cyclopentanediyl. These cyclic groups may be unsaturated. In particular, aromatic groups (arylene groups), for example phenylene, may be present in the hydrocarbon group. In turn, in the cyclic alkylene groups and the arylene groups, too, one or more hydrogen atoms may optionally be substituted by C₁₋₁₀-alkyl groups. In this way, an optionally branched hydrocarbon chain is formed. This hydrocarbon chain has a total of 0 to 100 carbon atoms, preferably 1 to 50, particularly preferably 2 to 25 carbon atoms.

The side chains, if present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

The hydrocarbon chain may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NH—, —C(═O)—, —NH—C(═O)—, —C(═O)—NH—, —NMe-, —NHNH—, —S(O)₂—NHNH—, —C(═O)—NHNH— and a 5- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from the group consisting of N, O and S, —S(═O)— or S(O)₂— (preferably

Further interrupting groups in G3 are preferably

According to the invention, L2 is preferably represented by the formulae: #¹—(NH)_(p)—(C═O)_(q)-G4-NH—#² or #¹—(NH)_(p)—(C═O)_(q)-G4-O—NH—#²

-   -   where     -   p is 0 or 1;     -   q is 0 or 1; and     -   G4 represents an optionally substituted alkyl or heteroalkyl         chain optionally wherein any carbon of the chain is substituted         with an alkoxy, hydroxyl, alkylcarbonyloxy, —S-alkyl, thiol,         —C(═O)—S-alkyl, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, amine,         —C(═O)—NH₂     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder,

Preferably, L2 is one of the groups below:

-   -   with Ry is —H, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —C(═O)—NH₂,         —NH₂,     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder.

Preferably Ry is —H or NH—C(═O)—Me.

Preferably, the linker corresponds to the formula below: § —(C═O)m-L1-L2-§ 517

-   -   where     -   m is 0 or 1;     -   § represents the bond to the active compound molecule and     -   § § represents the bond to the binder peptide or protein, and     -   L1 and L2 have the meaning given above.

The linkers mentioned above are especially preferred in conjugates of the formula (I) or (II) in which the linker couples by substitution of a hydrogen atom at R¹ or R³ or in combination with a cleavable linker SG1 at R⁴, i.e. R¹ represents -L-#1 or R³ represents -L-#1 or R⁴ represents -SG1-L-#1, where #1 represents the bond to the binder.

Preferred groups L1 in the formula § —(C═O)m-L1-L2-§ § above are those below, where r in each case independently of one another represents a number from 0 to 20, preferably from 0 to 15, particularly preferably from 1 to 20, especially preferably from 2 to 10:

Examples of conjugates having corresponding linkers have the following structures, where X1, X2, X3, Ry and L1 have the meanings given above, AK represents the binder, preferably an antibody conjugated to a glutamine side chain and n is 2 to 10, preferably 2 to 4 and also preferred 2 or 4. With particular preference, AK is a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof, in particular an anti-TWEAKR antibody or an antigen-binding fragment thereof or an anti-EGFR antibody or an antigen-binding fragment thereof. If the binder is an antibody it comprises an acceptor glutamine, preferentially in the constant region. Such acceptor glutamines can be introduced by mutations of suitable positions into glutamine (e.g. mutation N297Q, Kabat EU numbering) or by generation of deglycosylated or aglycosylated antibodies (e.g. by enzymatic deglycosylation by PNGase F or by mutation of N297X, Kabat EU numbering). In that later case of a deglycosylated or an aglycosylated antibody the glutamine Q295 (Kabat EU numbering) becomes an acceptor glutamine. Highly preferred is an antibody comprising a mutation N297A or N297Q (Kabat EU numbering). Particular preference is given to an anti-TWEAKR antibody which binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), in particular the anti-TWEAKR antibody TPP-2090, or the anti-Her2 antibody. All the antibodies described include aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

Preference according to the invention is furthermore given to the basic structure (i), (ii) or (iv), where SG1 or SG represents a group which can be cleaved by cathepsin and L1 and L2 have the meanings given above. Particular preference is given to the following groups:

-   -   —NH-Val-Ala-CONH— (hereby cleavage of the amide bond at the         C-terminal amide of alanine)     -   —NH-Val-Lys-CONH— (cleavage of the amide bond at the C-terminal         amide of lysine)     -   —NH-Val-Cit-CONH— (cleavage of the amide bond at the C-terminal         amide of citrulline)     -   —NH-Phe-Lys-CONH (cleavage of the amide bond at the C-terminal         amide of lysine)     -   —NH-Ala-Lys-CONH— (cleavage of the amide bond at the C-terminal         amide of lysine)     -   —NH-Ala-Cit-CONH— (cleavage of the amide bond at the C-terminal         amide of citrulline)

SG1 or SG is particularly preferably

where X represents —H or a C₁₋₁₀-alkyl group which may optionally be substituted by —NH—C(═O)—NH₂, —COOH, —OH, NH₂, —NH—CNNH₂ or sulphonic acid.

The table below gives examples of a linker moiety -SG1-L1- or -L1-SG-L1-, where SG1 and SG are groups which can be cleaved by cathepsin. The L1 group is highlighted in a box. However, these groups L1 can be replaced by one of the groups L1 given for formula § —(C═O)m-L1-L2-§ § above.

-SG1-L1- or -L1-SG-L1-

Examples of conjugates having basic structure (i) have the following structure, where X1, X2, X3, R1 and Ry have the meanings given above, AK represents the binder, preferably an antibody conjugated to a glutamine side chain and n is 2 to 10, preferably 2 to 4 and also preferred 2 or 4. Particularly preferably, AK is an anti-TWEAKR antibody, in particular an anti-TWEAKR antibody which binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), in particular the anti-TWEAKR antibody TPP-2090. All the antibodies described include aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

KSP Inhibitor-Linker-Intermediates and Preparation of the Conjugates

The conjugates according to the invention are prepared by initially providing the low-molecular weight KSP inhibitor with a linker. The intermediate obtained in this manner is then reacted with the binder (preferably an antibody) using transglutaminase.

Preferably, for site specific coupling to a glutamine side chain, one of the compounds below is reacted with the acceptor glutamine-containing binder such as an antibody using transglutaminase.

-   -   where X₁, X₂, X₃, SG, L1, R1 and Ry have the same meaning as         described above. In above-mentioned formulae, instead of a free         amino group (—NH₂), a group         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂—C(═O)—NH₂)—C(═O)—NH— may         be present (wherein R²¹, P2 and P3 have the same meaning as         defined above, e.g. with regard to formula (IIa)).

If the binder is an antibody it comprises an acceptor glutamine, preferentially in the constant region. Such acceptor glutamines can be introduced by mutations of suitable positions into glutamine (e.g. mutation N297Q, Kabat EU numbering) or by generation of deglycosylated or aglycosylated antibodies (e.g. by enzymatic deglycosylation by PNGase F or by mutation of N297X, Kabat EU numbering). In that later case of an deglycosylated or aglycosylated antibody the glutamine Q295 (Kabat EU numbering) becomes an acceptor glutamine. Highly preferred is an antibody comprising a mutation N297A or N297Q (Kabat EU numbering).

The compound may be employed, for example, in the form of its trifluoroacetic acid salt. For the reaction with the binder such as, for example, the antibody, the compound is preferably used in a 2 to 100 fold molar excess, more preferably 10 to 100 fold molar excess, even more preferably 50 to 100 fold molar excess, with respect to the binder.

For an intermediate coupling to a glutamine side chain, the reactions can be illustrated as follows: The other intermediates and other antibodies can be reacted correspondingly.

In accordance with the invention, this gives the following conjugates:

In the above formulae, X₁, X₂, X₃ and R1 have the same meaning as in formula (II), SG, Ry and L1 have the same meaning as described above. AK is an antibody coupled to a glutamine side chain. With particular preference, AK1 is anti-TWEAKR antibodies, in particular antibodies which bind specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), in particular the anti-TWEAKR antibody TPP-2090. All the antibodies described include aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

Particularly Preferred KSP-Inhibitor-Conjugates

Particularly preferred are the following conjugates, where AK3a, AK3b, AK3d, AK3e, represent the binder, preferably an antibody, and n represents 2 to 10, preferably 2 to 4, and also preferably 2 or 4. Preferred antibodies are those described below in the section Binders, particularly antibody TPP-2090-HC-N297A (particularly as AK3a), antibody TPP-2090-HC-N297Q (particularly as AK3b), Trastuzumab (TPP-7510) (equal to Trastuzumab-HC-N297A) (particularly as AK3d), and Trastuzumab (TPP-7511) (equal to Trastuzumab-HC-N297Q) (particularly as AK3e).

Definitions of Substituents

Alkyl

Alkyl represents a straight-chain or branched saturated monovalent hydrocarbon radical having generally 1 to 10, preferably 1 to 6 (C₁-C₆-alkyl), more preferably 1 to 4 (C₁-C₄-alkyl) and particularly preferably 1 to 3 (C₁-C₃-alkyl) carbon atoms.

Examples which may be mentioned as being preferred are:

methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, isopropyl-, isobutyl-, sec-butyl, tert-butyl-, isopentyl-, 2-methylbutyl-, 1-methylbutyl-, 1-ethylpropyl-, 1,2-dimethylpropyl, neopentyl-, 1,1-dimethylpropyl-, 4-methylpentyl-, 3-methylpentyl-, 2-methylpentyl-, 1-methylpentyl-, 2-ethylbutyl-, 1-ethylbutyl-, 3,3-dimethylbutyl-, 2,2-dimethylbutyl-, 1,1-dimethylbutyl-, 2,3-dimethylbutyl-, 1,3-dimethylbutyl-, 1,2-dimethylbutyl-.

Particular preference is given to a methyl, ethyl, propyl, isopropyl or tert-butyl radical.

Heteroalkyl

Heteroalkyl represents alkyl as defined under “Alkyl” above and is interrupted by —O—, —S—, —NH—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH—.

Cycloalkyl

Cycloalkyl represents a monocyclic saturated monovalent hydrocarbon radical having generally 3 to 10 (C₃-C₁₀-cycloalkyl), preferably 3 to 8

(C₃-C₈-cycloalkyl) and particularly preferably 3 to 7 (C3-C7-cycloalkyl) carbon atoms.

Examples of monocyclic cycloalkyl radicals which may be mentioned as being preferred are:

cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

Particular preference is given to a cyclopropyl, cyclopentyl or cyclohexyl radical.

Aralkyl

Aryl-C₁-C₆-alkyl- is understood to mean a group composed of an optionally substituted aryl radical and a C₁-C₆-alkyl group, and bonded to the rest of the molecule via the C₁-C₆-alkyl group. Here, the alkyl radical has the meanings given above under alkyl.

Examples which may be mentioned include benzyl, phenylethyl, phenylpropyl, phenylpentyl, with benzyl being preferred.

Alkoxy

Alkoxy represents a straight-chain or branched saturated alkylether radical of the formula —O-alkyl having generally 1 to 6 (C₁-C₆-alkoxy), preferably 1 to 4 (C₁-C₄-alkoxy) and particularly preferably 1 to 3 (C₁-C₃-alkoxy) carbon atoms.

Examples which may be mentioned as being preferred are:

methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentyloxy and n-hexyloxy.

Aralkoxy

Aralkoxy is understood to mean a group composed of an optionally substituted aryl radical and a C₁-C₆-alkoxy group, and bonded to the rest of the molecule via the C₁-C₆-alkox group. Here, the alkoxy radical has the meanings as defined above.

Examples which may be mentioned include benzyloxy, phenylethyloxy, phenylpropyloxy, phenylpentyloxy, with benzyloxy being preferred.

Alkoxyalkyl

Alkoxyalkyl represents an alkyl radical substituted by alkoxy, for example C₁-C₆-alkoxy-C₁-C₆-alkyl- or C₁-C₃-alkoxy-C₁-C₃-alkyl-.

Here, C₁-C₆-alkoxy-C₁-C₆-alkyl- means that the alkoxyalkyl group is attached via the alkyl moiety to the remainder of the molecule.

Heteroatoms

Heteroatoms are understood to mean oxygen, nitrogen or sulphur atoms.

Aryl

Aryl represents a monovalent mono- or bicyclic aromatic ring system which consists of carbon atoms. Examples are naphthyl- and phenyl-; preference is given to phenyl- or a phenyl radical.

Heteroaryl

Heteroaryl represents a monovalent mono- or bicyclic aromatic ring system having one, two, three or four heteroatoms which may be identical or different. The heteroatoms may be nitrogen atoms, oxygen atoms or sulphur atoms. The binding valency can be at any aromatic carbon atom or at a nitrogen atom.

A monocyclic heteroaryl radical in accordance with the present invention has 5 or 6 ring atoms. Preference is given to heteroaryl radicals having one or two heteroatoms. Here, particular preference is given to one or two nitrogen atoms.

Heteroaryl radicals having 5 ring atoms include, for example, the rings:

thienyl, thiazolyl, furyl, pyrrolyl, oxazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, tetrazolyl and thiadiazolyl.

Heteroaryl radicals having 6 ring atoms include, for example, the rings:

pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.

A bicyclic heteroaryl radical in accordance with the present invention has 9 or 10 ring atoms.

Heteroaryl radicals having 9 ring atoms include, for example, the rings:

phthalidyl, thiophthalidyl, indolyl, isoindolyl, indazolyl, benzothiazolyl, benzofuryl, benzothienyl, benzimidazolyl, benzoxazolyl, azocinyl, indolizinyl, purinyl, indolinyl.

Heteroaryl radicals having 10 ring atoms include, for example, the rings:

isochinolinyl, quinolinyl, quinolizinyl, quinazolinyl, quinoxalinyl, cinnolinyl, phthalazinyl, 1,7- and 1,8-naphthyridinyl, pteridinyl, chromanyl.

Heteroaryl further has the meaning of partially saturated bicyclic aryl and partially saturated bicyclic heteroaryl.

Partially Saturated Bicyclic Aryl and Partially Saturated Bicyclic Heteroaryl

A partially saturated bicyclic aryl radical or heteroaryl radical represents a bicyclic group consisting of a phenyl radical or a monocyclic 5- or 6-membered heteroaryl radical which is condensed via two directly adjacent ring atoms in each case to an aliphatic cyclic radical having 4 to 7 ring atoms which may optionally contain one or two heteroatoms which may be identical or different. The heteroatoms may be nitrogen atoms, oxygen atoms or sulphur atoms.

Partially saturated bicyclic aryl radicals include, for example, the groups:

tetrahydronaphthyl, 2,3-dihydro-1,4-benzodioxinyl-, 2,3-dihydro-1-benzofuranyl- and 1,3-benzodioxolyl-

Partially saturated bicyclic heteroaryl radicals include, for example, the groups:

5,6,7,8-tetrahydroquinolinyl- and 5,6,7,8-tetrahydroisoquinolinyl-.

Heterocycloalkyl

Heterocycloalkyl stands for monocyclic heterocyclyl, heterospirocycloalkyl and bridged heterocycloalkyl.

Monocyclic Heterocyclyl

Monocyclic heterocyclyl- means a non-aromatic monocyclic ring system having one, two or three heteroatoms which may be identical or different. The heteroatoms may be nitrogen atoms, oxygen atoms or sulphur atoms.

A monocyclic heterocyclyl ring according to the present invention may have 3 to 8, preferably 4 to 7, particularly preferably 5 or 6 ring atoms.

By way of example and with preference, the following may be mentioned for monocyclic heterocyclyl radicals having 3 ring atoms:

aziridinyl-.

By way of example and with preference, the following may be mentioned for monocyclic heterocyclyl radicals having 4 ring atoms:

azetidinyl-, oxetanyl-.

By way of example and with preference, the following may be mentioned for monocyclic heterocyclyl radicals having 5 ring atoms:

pyrrolidinyl-, imidazolidinyl-, pyrazolidinyl-, pyrrolinyl-, dioxolanyl- and tetrahydrofuranyl-.

By way of example and with preference, the following may be mentioned for monocyclic heterocyclyl radicals having 6 ring atoms:

piperidinyl-, piperazinyl-, morpholinyl-, dioxanyl-, tetrahydropyranyl- and thiomorpholinyl-.

By way of example and with preference, the following may be mentioned for monocyclic heterocyclyl radicals having 7 ring atoms:

azepanyl-, oxepanyl-, 1,3-diazepanyl-, 1,4-diazepanyl-.

By way of example and with preference, the following may be mentioned for monocyclic heterocyclyl radicals having 8 ring atoms:

oxocanyl-, azocanyl-.

From among the monocyclic heterocyclyl radicals, preference is given to 4- to 7-membered saturated heterocyclyl radicals having up to two heteroatoms from the group consisting of O, N and S.

Particular preference is given to morpholinyl-, piperidinyl- and pyrrolidinyl-.

Spirocycloalkyl and Heterospirocycloalkyl

C₅-C₁₂-Spirocycloalkyl or C₅-C₁₂-heterospirocycloalkyl where one, two, three or four carbon atoms are replaced by heteroatoms as defined above in any combination is understood to mean a fusion of two saturated ring systems which share one common atom. Examples are spiro[2.2]pentyl, spiro[2.3]hexyl, azaspiro[2.3]hexyl, spiro[3.3]heptyl, azaspiro[3.3]heptyl, oxaazaspiro[3.3]heptyl, thiaazaspiro[3.3]heptyl, oxaspiro[3.3]heptyl, oxazaspiro[3.5]nonyl, oxazaspirop[3.4]octyl, oxazaspiro[5.5]undecyl, diazaspiro[3.3]heptyl, thiazaspiro[3.3]heptyl, thiazaspiro[3.4]octyl, azaspiro[5.5]decyl, and the further homologous spiro[3.4], spiro[4.4], spiro[5.5], spiro[6.6], spiro[2.4], spiro[2.5], spiro[2.6], spiro[3.5], spiro[3.6], spiro[4.5], spiro[4.6] and spiro[5.6] systems including the variants modified by heteroatoms as per the definition. Preference is given to C6-C10-heterospirocycloalkyl-, by way of example and with particular preference 2-azaspiro[3.3]heptyl-,

1-thia-6-azaspiro[3.3]heptyl-, 2-thia-6-azaspiro[3.3]heptyl-, 2-oxa-6-azaspiro[3.3]heptyl-, 2,6-diazaspiro[3.3]heptyl-, 2-oxa-6-azaspiro[3.4]octyl-, 2-oxa-6-azaspiro[3.5]nonyl-, 2-oxa-7-azaspiro[3.5]nonyl-, 8-azaspiro[4.5]decyl-, 2,8-diazaspiro[4.5]decyl-, 3-oxa-1,8-diazaspiro[4.5]decyl-. Bicycloalkyl and Heterobicycloalkyl

C₆-C₁₂-Bicycloalkyl or C₆-C₁₂-heterobicycloalkyl where one, two, three or four carbon atoms are replaced by heteroatoms as defined above in any combination is understood to mean a fusion of two saturated ring systems which share two directly adjacent atoms. Examples are radicals derived from bicyclo[2.2.0]hexyl-, bicyclo[3.3.0]octyl-, bicyclo[4.4.0]decyl-, bicyclo[5.4.0]undecyl-, bicyclo[3.2.0]heptyl-, bicyclo[4.2.0]octyl-, bicyclo[5.2.0]nonyl-, bicyclo[6.2.0]decyl-, bicyclo[4.3.0]nonyl-, bicyclo[5.3.0]decyl-, bicyclo[6.3.0]undecyl- and bicyclo[5.4.0]undecyl-, including the variants modified by heteroatoms, for example azabicyclo[3.3.0]octyl-, azabicyclo[4.3.0]nonyl-, diazabicyclo[4.3.0]nonyl-, oxazabicyclo[4.3.0]nonyl-, thiazabicyclo[4.3.0]nonyl- or azabicyclo[4.4.0]decyl-, and the further possible combinations as per the definition. Preference is given to C₆-C₁₀-heterobicycloalkyl-, by way of example and with particular preference perhydrocyclopenta[c]pyrrolyl-, perhydrofuro[3,2-c]pyridinyl-, perhydropyrrolo[1,2-a]pyrazinyl-, perhydropyrrolo[3,4-c]pyrrolyl-.

Preferred examples of C₆-C₁₂-bicycloalkyl- are perhydronaphthalenyl- (decalinyl-), perhydrobenzoannulenyl-, perhydroazulenyl-, perhydroindanyl-, perhydropentalenyl-.

Bridged Cycloalkyl and Bridged Heterocycloalkyl

A bridged C₆-C₁₂ ring system such as bridged C₆-C₁₂-cycloalkyl- or bridged C₆-C₁₂-heterocycloalkyl- is understood to mean a fusion of at least two saturated rings which share two atoms that are not directly adjacent to one another. This may give rise either to a bridged carbocycle (bridged cycloalkyl-) or to a bridged heterocycle (bridged heterocycloalkyl-) where one, two, three or four carbon atoms are replaced by heteroatoms as defined above in any combination. Examples are bicyclo[2.2.1]heptyl-, azabicyclo[2.2.1]heptyl-, oxazabicyclo[2.2.1]heptyl-, thiazabicyclo[2.2.1]heptyl-, diazabicyclo[2.2.1]heptyl-, bicyclo[2.2.2]octyl-, azabicyclo[2.2.2]octyl-, diazabicyclo[2.2.2]octyl-, oxazabicyclo[2.2.2]octyl-, thiazabicyclo[2.2.2]octyl-, bicyclo[3.2.1]octyl-, azabicyclo[3.2.1]octyl-, diazabicyclo[3.2.1]octyl-, oxazabicyclo[3.2.1]octyl-, thiazabicyclo[3.2.1]octyl-, bicyclo[3.3.1]nonyl-, azabicyclo[3.3.1]nonyl-, diazabicyclo[3.3.1]nonyl-oxazabicyclo[3.3.1]nonyl-, thiazabicyclo[3.3.1]nonyl-, bicyclo[4.2.1]nonyl-, azabicyclo[4.2.1]nonyl-, diazabicyclo[4.2.1]nonyl-, oxazabicyclo[4.2.1]nonyl-, thiazabicyclo[4.2.1]nonyl-, bicyclo[3.3.2]decyl-, azabicyclo[3.3.2]decyl-, diazabicyclo[3.3.2]decyl-, oxazabicyclo[3.3.2]decyl-, thiazabicyclo[3.3.2]decyl- or azabicyclo[4.2.2]decyl- and the further possible combinations according to the definition. Preference is given to bridged C₆-C₁₀-heterocycloalkyl-, by way of example and with particular preference

2-azabicyclo[2.2.1]heptyl-, 2,5-diazabicyclo[2.2.1]heptyl-, 2-oxa-5-azabicyclo[2.2.1]heptyl-, 8-azabicyclo[3.2.1]octyl-, 8-oxa-3-azabicyclo[3.2.1]octyl-, 3,9-diazabicyclo[4.2.1]nonyl-.

Halogenated Alkyl Groups

A halogenated alkyl group represents an alkyl radical having at least one halogen substituent.

A halo-C₁-C₆-alkyl radical is an alkyl radical having 1-6 carbon atoms and at least one halogen substituent. If a plurality of halogen substituents is present, these may also be different from one another. Preference is given to fluoro-C₁-C₆-alkyl, fluoro-C₁-C₄-alkyl and fluoro-C₁-C₃-alkyl radicals. Examples which may be mentioned as being likewise preferred are:

the trifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 4,4,5,5,5-pentafluoropentyl or 3,3,4,4,5,5,5-heptafluoropentyl group.

Preference is given to perfluorinated alkyl radicals such as trifluoromethyl or pentafluoroethyl.

Further Definitions

The term “transglutaminase”, used interchangeably with “TGase” or “TG”, refers to an enzyme capable of cross-linking proteins through an acyl-transfer reaction between the γ-carboxamide group of peptide-bound glutamine and the ε-amino group of a lysine or a structurally related primary amine such as amino pentyl group, e.g. a peptide-bound lysine, resulting in a 8-(y-glutamyl) lysine isopeptide bond. TGases include, inter glia, bacterial transglutaminase (BIG) such as the enzyme having EC reference EC 2.3.2.13 (protein-glutamine-y-glutamyltransferase).

The term “acceptor glutamine”, when referring to an amino acid residue of an antibody, means a glutamine residue that, under suitable conditions, is recognized by a TGase and can be cross-linked by a TGase through a reaction between the glutamine and a lysine or a structurally related primary amine such as amino pentyl group. Optionally the acceptor glutamine is a surface-exposed glutamine.

By “amino acid modification” or by “mutation” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. The preferred amino acid modification herein is a substitution.

By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution Y50W refers to a variant of a parent polypeptide, in which the tyrosine at position 50 is replaced with tryptophan. A “variant” of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or “parent” polypeptide. The polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.

The term “site-specific conjugate” refers to a conjugate of a binder, preferably an antibody, and a moiety, preferably a linker-drug-moiety, wherein the binder is functionalized at one or more defined positions, preferably glutamine residues, of the binder. Transglutaminases (TGase) including bacterial transglutaminase (BTG) (EC 2.3.2.13) display strict specificity in recognition of glutamine protein substrates and can catalyze a “site-specific conjugation”.

The term “homogeneous conjugate” or “homogeneous ADC” refers to a composition of site specific conjugates wherein at least 60, 70, 80 or 90% of the binders have the same number of conjugated moieties per binder. In case of an antibody the number of conjugated moieties per antibody might be an even number, preferentially 2 or 4.

Binders

In the broadest sense, the term “binder” is understood to mean a molecule which binds to a target molecule present at a certain target cell population to be addressed by the binder/active compound conjugate. The term binder is to be understood in its broadest meaning and also comprises, for example, lectins, proteins capable of binding to certain sugar chains, and phospholipid-binding proteins. Such binders include, for example, high-molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptidic (e.g. aptamers (U.S. Pat. No. 5,270,163) review by Keefe A D., et al., Nat. Rev. Drug Discov. 2010; 9:537-550), or vitamins) and all other cell-binding molecules or substances. Binding proteins are, for example, antibodies including aglycosylated variants, and antibody fragments or antibody mimetics such as, for example, affibodies, adnectins, anticalins, DARPins, avimers, nanobodies (review by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S. D. et al., Curr. Opinion in Pharmacology 2008; 8:608-617). Binding peptides are, for example, ligands of a ligand/receptor pair such as, for example, VEGF of the ligand/receptor pair VEGF/KDR, such as transferrin of the ligand/receptor pair transferrin/transferrin receptor or cytokine/cytokine receptor, such as TNFalpha of the ligand/receptor pair TNFalpha/TNFalpha receptor.

The “binder” comprises an acceptor glutamine residue which can be functionalized by a transglutaminase (TGase) including bacterial transglutaminase (BTG) (EC 2.3.2.13). This acceptor glutamine is either naturally occurring without any alteration of the binder or has been generated. An acceptor glutamine might be generated via insertion of a glutamine residue at a suitable position (e.g. via fusion to a tag comprising an acceptor glutamine), mutation of a suitable position into a glutamine residue, mutation of an amino acid residue wherein the mutation has the effect that a naturally occurring glutamine residue formerly not recognized by an TGase becomes an acceptor glutamine, or changing the post translational modification (e.g. glycosylation) wherein the change has the effect that a naturally occurring glutamine residue formerly not recognized by an TGase becomes an acceptor glutamine. If the binder is an antibody it comprises an acceptor glutamine, preferentially in the constant region. Such acceptor glutamines can be introduced by mutations of suitable positions into glutamine (e.g. mutation N297Q, Kabat EU numbering) or by generation of deglycosylated or aglycosylated antibodies (e.g. by enzymatic deglycosylation by PNGase F or by mutation of N297X, Kabat EU numbering). In that later case of a deglycosylated or an aglycosylated antibody the glutamine Q295 (Kabat EU numbering) becomes an acceptor glutamine. Highly preferred is an antibody comprising a mutation N297A or N297Q (Kabat EU numbering).

The term “aglycosyl antibody” or “aglycosylated antibody” or “deglycosylated antibody” herein is used to define an antibody or an antibody derivative which comprises an Fc region lacking the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region. Aglycosyl antibodies can for example be prepared by mutation of the heavy chain glycosylation site of N297 (using Kabat EU numbering) or by expression the antibodies in expression systems lacking glycosylation. Methods for enzymatic deglycosylation of antibodies are well known in the art (e.g. Winkelhake & Nicolson (1976), J Biol Chem. 251(4):1074-80). Deglycosylated antibodies may e.g. be prepared by enzymatic deglycosylation using e.g. PNGase F. In one embodiment of this invention, aglycosyl antibodies may be prepared by expression the antibodies in a prokaryotic host. Suitable prokaryotic hosts for include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In another embodiment of this invention, aglycosyl antibodies may be achieved using mammalian expression systems together with the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983), Proc Natl Acad Sci USA, 80(21):6632-6). That is, the modification is the prevention of glycosylation at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody.

The literature also discloses various options of homogenous site specific covalent coupling (conjugation) of organic molecules to antibodies. Preference according to the invention is given to the conjugation of the toxophores to the antibody via two or four acceptor glutamine residues of the antibody.

A “target molecule” in the broadest sense is understood to mean a molecule which is present in the target cell population and which may be a protein (for example a receptor of a growth factor) or a non-peptidic molecule (for example a sugar or phospholipid). It is preferably a receptor or an antigen.

The term “extracellular” target molecule describes a target molecule, attached to the cell, which is located at the outside of a cell, or the part of a target molecule which is located at the outside of a cell, i.e. a binder may bind on an intact cell to its extracellular target molecule. An extracellular target molecule may be anchored in the cell membrane or be a component of the cell membrane. The person skilled in the art is aware of methods for identifying extracellular target molecules. For proteins, this may be by determining the transmembrane domain(s) and the orientation of the protein in the membrane. These data are usually deposited in protein databases (e.g. SwissProt).

The term “cancer target molecule” describes a target molecule which is more abundantly present on one or more cancer cell species than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell species compared with non-cancer cells of the same tissue type, where selectively describes an at least two-fold enrichment on cancer cells compared to non-cancer cells of the same tissue type (a “selective cancer target molecule”). The use of cancer target molecules allows the selective therapy of cancer cells using the conjugates according to the invention.

The binder can be attached to the linker via a bond. Attachment of the binder can be via a carbonyl function of a glutamine side chain. Preferably such glutamine residues are recognized as substrates by bacterial transglutaminase. Glutamine residues according to the invention may be present in the natural binder or are introduced by methods of molecular biology, e.g. by deglycosylation of the antibody by PNGaseF or by introduction of mutations. According to the invention, the attachment of the binder to the toxophor has only a minor effect on the binding activity of the binder with respect to the target molecule. In a preferred embodiment, the attachment has no effect on the binding affinity and specificity of the binder with respect to the target molecule.

In accordance with the present invention, the term “antibody” is to be understood in its broadest meaning and comprises immunoglobulin molecules, for example intact or modified monoclonal antibodies, aglycosylated antibodies, polyclonal antibodies or multispecific antibodies (e.g. bispecific antibodies). An immunoglobulin molecule preferably comprises a molecule having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains) which are typically linked by disulphide bridges. Each heavy chain comprises a variable domain of the heavy chain (abbreviated VH) and a constant domain of the heavy chain. The constant domain of the heavy chain may, for example, comprise three domains CH1, CH2 and CH3. Each light chain comprises a variable domain (abbreviated VL) and a constant domain. The constant domain of the light chain comprises a domain (abbreviated CL). The VH and VL domains may be subdivided further into regions having hypervariability, also referred to as complementarity determining regions (abbreviated CDR) and regions having low sequence variability (framework region, abbreviated FR). Typically, each VH and VL region is composed of three CDRs and up to four FRs. For example from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody may be obtained from any suitable species, e.g. rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin. An antibody may, for example, be human, humanized or chimeric.

The term “monoclonal” antibody refers to antibodies obtained from a population of substantially homogeneous antibodies, i.e. individual antibodies of the population are identical except for naturally occurring mutations, of which there may be a small number. Monoclonal antibodies recognize a single antigenic binding site with high specificity. The term monoclonal antibody does not refer to a particular preparation process.

The term “intact” antibody refers to antibodies comprising both an antigen-binding domain and the constant domain of the light and heavy chain. The constant domain may be a naturally occurring domain or a variant thereof having a number of modified amino acid positions and can be also aglycosylated.

The term “modified intact” antibody refers to intact antibodies fused via their amino terminus or carboxy terminus by means of a covalent bond (e.g. a peptide bond) with a further polypeptide or protein not originating from an antibody. Furthermore, antibodies may be modified such that, at defined positions, reactive cysteines are introduced to facilitate coupling to a toxophor (see Junutula et al. Nat Biotechnol. 2008 August; 26(8):925-32).

The term “human” antibody refers to antibodies which can be obtained from a human or which are synthetic human antibodies. A “synthetic” human antibody is an antibody which is partially or entirely obtainable in silico from synthetic sequences based on the analysis of human antibody sequences. A human antibody can be encoded, for example, by a nucleic acid isolated from a library of antibody sequences of human origin. An example of such an antibody can be found in Söderlind et al., Nature Biotech. 2000, 18:853-856. Such “human” and “synthetic” antibodies also include aglycosylated variants generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

The term “humanized” or “chimeric” antibody describes antibodies consisting of a non-human and a human portion of the sequence. In these antibodies, part of the sequences of the human immunoglobulin (recipient) are replaced by sequence portions of a non-human immunoglobulin (donor). In many cases, the donor is a murine immunoglobulin. In the case of humanized antibodies, amino acids of the CDR of the recipient are replaced by amino acids of the donor. Sometimes, amino acids of the framework, too, are replaced by corresponding amino acids of the donor. In some cases the humanized antibody contains amino acids present neither in the recipient nor in the donor, which were introduced during the optimization of the antibody. In the case of chimeric antibodies, the variable domains of the donor immunoglobulin are fused with the constant regions of a human antibody. Such “humanized” and “chimeric” antibodies also include aglycosylated variants generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

The term complementarity determining region (CDR) as used herein refers to those amino acids of a variable antibody domain which are required for binding to the antigen. Typically, each variable region has three CDR regions referred to as CDR1, CDR2 and CDR3. Each CDR region may embrace amino acids according to the definition of Kabat and/or amino acids of a hypervariable loop defined according to Chotia. The definition according to Kabat comprises, for example, the region from about amino acid position 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) of the variable light chain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) of the variable heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The definition according to Chotia comprises, for example, the region from about amino acid position 26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) of the variable light chain and 26-32 (CDR1), 53-55 (CDR2) and 96-101 (CDR3) of the variable heavy chain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some cases, a CDR may comprise amino acids from a CDR region defined according to Kabat and Chotia.

Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies may be categorized into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be divided into further subclasses. (Isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of the heavy chain, which correspond to the different classes, are referred to as [alpha/α], [delta/δ], [epsilon/ε], [gamma/γ] and [my/μ]. Both the three-dimensional structure and the subunit structure of antibodies are known.

The term “functional fragment” or “antigen-binding antibody fragment” of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (e.g. the variable domains of an IgG) which still comprise the antigen binding domains of the antibody/immunoglobulin. The “antigen binding domain” of an antibody typically comprises one or more hypervariable regions of an antibody, for example the CDR, CDR2 and/or CDR3 region. However, the “framework” or “skeleton” region of an antibody may also play a role during binding of the antibody to the antigen. The framework region forms the skeleton of the CDRs. Preferably, the antigen binding domain comprises at least amino acids 4 to 103 of the variable light chain and amino acids 5 to 109 of the variable heavy chain, more preferably amino acids 3 to 107 of the variable light chain and 4 to 111 of the variable heavy chain, particularly preferably the complete variable light and heavy chains, i.e. amino acids 1-109 of the VL and 1 to 113 of the VH (numbering according to WO97/08320).

“Functional fragments” or “antigen-binding antibody fragments” of the invention encompass, non-conclusively, Fab, Fab′, F(ab′)2 and Fv fragments, diabodies, Single Domain Antibodies (DAbs), linear antibodies, individual chains of antibodies (single-chain Fv, abbreviated to scFv); and multispecific antibodies, such as bi and tri-specific antibodies, for example, formed from antibody fragments C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag. Antibodies other than “multispecific” or “multifunctional” antibodies are those having identical binding sites. Multispecific antibodies may be specific for different epitopes of an antigen or may be specific for epitopes of more than one antigen (see, for example WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60 69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol. 148: 1547 1553). An F(ab′)2 or Fab molecule may be constructed such that the number of intermolecular disulphide interactions occurring between the Ch1 and the CL domains can be reduced or else completely prevented.

“Epitopes” refer to protein determinants capable of binding specifically to an immunoglobulin or T cell receptors. Epitopic determinants usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains or combinations thereof, and usually have specific 3-dimensional structural properties and also specific charge properties.

“Functional fragments” or “antigen-binding antibody fragments” may be fused with another polypeptide or protein, not originating from an antibody, via the amino terminus or carboxyl terminus thereof, by means of a covalent bond (e.g. a peptide linkage). Furthermore, antibodies and antigen-binding fragments may be modified by introducing reactive cysteines at defined locations, in order to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol. 2008 August; 26(8):925-32).

Polyclonal antibodies can be prepared by methods known to a person of ordinary skill in the art. Monoclonal antibodies may be prepared by methods known to a person of ordinary skill in the art (Köhler and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies may be prepared by methods known to a person of ordinary skill in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et al. U.S. Pat. No. 4,816,567 or Boss et al. U.S. Pat. No. 4,816,397).

A person of ordinary skill in the art is aware of diverse methods for preparing human antibodies and fragments thereof, such as, for example, by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol. 1995; 13(1):65-93) or Phage Display Technologien (Clackson et al., Nature. 1991 Aug. 15; 352(6336):624-8). Antibodies of the invention may be obtained from recombinant antibody libraries consisting for example of the amino acid sequences of a multiplicity of antibodies compiled from a large number of healthy volunteers. Antibodies may also be produced by means of known recombinant DNA technologies. The nucleic acid sequence of an antibody can be obtained by routine sequencing or is available from publically accessible databases.

An “isolated” antibody or binder has been purified to remove other constituents of the cell. Contaminating constituents of a cell which may interfere with a diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptidic or non-peptidic constituents of a cell. A preferred antibody or binder is one which has been purified to an extent of more than 95% by weight, relative to the antibody or binder (determined for example by Lowry method, UV-Vis spectroscopy or by SDS capillary gel electrophoresis). Moreover an antibody which has been purified to such an extent that it is possible to determine at least 15 amino acids of the amino terminus or of an internal amino acid sequence, or which has been purified to homogeneity, the homogeneity being determined by SDS-PAGE under reducing or non-reducing conditions (detection may be determined by means of Coomassie Blau staining or preferably by silver coloration). However, an antibody is normally prepared by one or more purification steps.

The term “specific binding” or “binds specifically” refers to an antibody or binder which binds to a predetermined antigen/target molecule. Specific binding of an antibody or binder typically describes an antibody or binder having an affinity of at least 10⁻⁷ M (as Kd value; i.e. preferably those with smaller Kd values than 10⁻⁷ M), with the antibody or binder having an at least two times higher affinity for the predetermined antigen/target molecule than for a non-specific antigen/target molecule (e.g. bovine serum albumin, or casein) which is not the predetermined antigen/target molecule or a closely related antigen/target molecule. The antibodies preferably have an affinity of at least 10⁻⁷ M (as Kd value; in other words preferably those with smaller Kd values than 10⁻⁷ M), preferably of at least 10⁻⁸ M, more preferably in the range from 10⁻⁹ M to 10⁻¹¹ M. The Kd values may be determined, for example, by means of surface plasmon resonance spectroscopy.

The antibody-drug conjugates of the invention likewise exhibit affinities in these ranges. The affinity is preferably not substantially affected by the conjugation of the drugs (in general, the affinity is reduced by less than one order of magnitude, in other words, for example, at most from 10⁻⁸ M to 10⁻⁷ M).

The antibodies used in accordance with the invention are also notable preferably for a high selectivity. A high selectivity exists when the antibody of the invention exhibits an affinity for the target protein which is better by a factor of at least 2, preferably by a factor of 5 or more preferably by a factor of 10, than for an independent other antigen, e.g. human serum albumin (the affinity may be determined, for example, by means of surface plasmon resonance spectroscopy).

Furthermore, the antibodies of the invention that are used are preferably cross-reactive. In order to be able to facilitate and better interpret preclinical studies, for example toxicological or activity studies (e.g. in xenograft mice), it is advantageous if the antibody used in accordance with the invention not only binds the human target protein but also binds the species target protein in the species used for the studies. In one embodiment the antibody used in accordance with the invention, in addition to the human target protein, is cross-reactive to the target protein of at least one further species. For toxicological and activity studies it is preferred to use species of the families of rodents, dogs and non-human primates. Preferred rodent species are mouse and rat. Preferred non-human primates are rhesus monkeys, chimpanzees and long-tailed macaques.

In one embodiment the antibody used in accordance with the invention, in addition to the human target protein, is cross-reactive to the target protein of at least one further species selected from the group of species consisting of mouse, rat and long-tailed macaque (Macaca fascicularis). Especially preferred are antibodies used in accordance with the invention which in addition to the human target protein are at least cross-reactive to the mouse target protein. Preference is given to cross-reactive antibodies whose affinity for the target protein of the further non-human species differs by a factor of not more than 50, more particularly by a factor of not more than ten, from the affinity for the human target protein.

Antibodies Directed Against a Cancer Target Molecule

The target molecule towards which the binder, for example an antibody or an antigen-binding fragment thereof, is directed is preferably a cancer target molecule. The term “cancer target molecule” describes a target molecule which is more abundantly present on one or more cancer cell species than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell species compared with non-cancer cells of the same tissue type, where selectively describes an at least two-fold enrichment on cancer cells compared to non-cancer cells of the same tissue type (a “selective cancer target molecule”). The use of cancer target molecules allows the selective therapy of cancer cells using the conjugates according to the invention.

Antibodies which are specific against an antigen, for example cancer cell antigen, can be prepared by a person of ordinary skill in the art by means of methods with which he or she is familiar (such as recombinant expression, for example) or may be acquired commercially (as for example from Merck KGaA, Germany). Examples of known commercially available antibodies in cancer therapy are Erbitux® (cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche) and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1kappa type which in a cell-based assay (Kd=5 nM) binds the extracellular domains of the human epidermal growth receptor with high affinity. The antibody is produced recombinantly in CHO cells. All these antibodies can be also prepared as aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid.

In a preferred embodiment, the target molecule is a selective cancer target molecule.

In a particularly preferred embodiment, the target molecule is a protein.

In one embodiment, the target molecule is an extracellular target molecule. In a preferred embodiment, the extracellular target molecule is a protein.

Cancer target molecules are known to those skilled in the art. Examples of these are listed below.

Examples of cancer target molecules are:

(1) EGF receptor (NCBI reference sequence NP_005219.2), SEQ ID NO: 213 (1210 amino acids):

>gi|29725609|ref|NP_005219.2|EGFR  receptor precursor [Homo sapiens] MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLS LQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIP LENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRF SNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCW GAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLV CRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYV VTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLS INATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKE ITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGL RSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFV ENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVM GENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGM VGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPN QALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREA TSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLD YVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQH VKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSY GVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKC WMIDADSRPKFRELHEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRAL MDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACID RNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRP AGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTF DSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVA PQSSEFIGA

The extracellular domain is marked by underlining.

(2) mesothelin (SwissProt reference Q13421-3), SEQ ID NO: 214 (622 amino acids):

>sp|Q13421-3|MSLN_HUMAN isoform 2  of mesothelin OS=Homo sapiens GN=MSLN MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDG VLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQ LRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVD LLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAES AEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRG LLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLD VLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLE VNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELS SVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFL GGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGL KAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGT PCLLGPGPVLTVLALLLASTLA where mesothelin is encoded by amino acids 296-598. Amino acids 37-286 are coding for the megakaryocyte-potentiating factor. Mesothelin is anchored in the cell membrane via a GPI anchor and is localized extracellularly. (3) carboanhydrase IX (SwissProt reference Q16790), SEQ ID NO: 215 (459 amino acids):

>sp|Q16790|CAH9_HUMAN carbonic  anhydrase 9 OS=Homo sapiens GN=CA9 PE=1 SV=2 MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHPQRLPRMQEDSPLG GGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKP KSEEEGSLKLEDLPTVEAPGDPQEPQNNAHRDKEGDDQSHWRYGGDPPWP RVSPACAGRFQSPVDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGH SVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHTVEGHRFPAEI HVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIA EEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVM LSAKQLHTLSDTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRA AEPVQLNSCLAAGDILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYR PAEVAETGA

The extracellular domain is marked by underlining.

(4) C4.4a (NCBI reference sequence NP_055215.2; synonym LYPD3), SEQ ID NO: 216 (346 amino acids):

>gi|93004088|ref|NP_055215.2|ly6/PLAUR domain- containing protein 3-precursor [Homo sapiens] MDPARKAGAQAMIWTAGWLLLLLLRGGAQALECYSCVQKADDGCSPNKMK TVKCAPGVDVCTEAVGAVETIHGQFSLAVRGCGSGLPGKNDRGLDLHGLL AFIQLQQCAQDRCNAKLNLTSRALDPAGNESAYPPNGVECYSCVGLSREA CQGTSPPVVSCYNASDHVYKGCFDGNVTLTAANVTVSLPVRGCVQDEFCT RDGVTGPGFTLSGSCCQGSRCNSDLRNKTYFSPRIPPLVRLPPPEPTTVA STTSVTTSTSAPVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEPRLTGGA AGHQDRSNSGQYPAKGGPQQPHNKGCVAPTAGLAALLLAVAAGVLL

The mature extracellular domain is marked by underlining.

(5) CD52 (NCBI reference sequence NP_001794.2), SEQ ID NO: 217

>gi|68342030|ref|NP_001794.2|  CAMPATH-1 antigen-precursor [Homo sapiens] MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFV ANAIIHLFCFS (6) Her2 (NCBI reference sequence NP_004439.2), SEQ ID NO: 218

>gi|54792096|ref|NP_004439.2| receptor tyrosine- protein kinase erbB-2 isoform a [Homo sapiens] MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLY QGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLR IVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILK GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCK GSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHS DCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHL REVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVF ETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGI SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRP EDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGL PREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASP LTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPL TPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPV AIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQL MPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARN VLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTID VYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPL DSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSS STRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQS LPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPP SPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLG LDVPV (7) CD20 (NCBI reference sequence NP_068769.2), SEQ ID NO: 219

>gi|23110987|ref|NP_068769.2| B- lymphocyte antigen CD20 [Homo sapiens] MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESK TLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSL LAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKME SLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIF AFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLT ETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP (8) the lymphocyte activation antigen CD30 (SwissProt ID P28908), SEQ ID NO: 220

>gi|68348711|ref|NP_001234.2|  tumor necrosis factor receptor superfamily  member 8 isoform 1-precursor [Homo sapiens] MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPM GLFPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVTCSRDDLVEKTPCAW NSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFPGTAQKNTVCE PASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRL AQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYL DEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSRARC VPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGEAPASTSP TQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVG SSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSG ASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDL PEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEE ELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK (9) the lymphocyte adhesion molecule CD22 (SwissProt ID P20273), SEQ ID NO: 221

>gi|157168355|ref|NP_001762.2| B-cell receptor CD22 isoform 1-precursor [Homo sapiens] MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALD GDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNK NCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQLPPE IQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFT RSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTP SDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVT KDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVE FLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAEN ILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPS VTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASPVALN VQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLL GKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM SPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVK VQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILI LAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLG CYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALH KRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH (10) the myloid cell surface antigen CD33 (SwissProt ID P20138), SEQ ID NO: 222

>gi|130979981|ref|NP_001763.3| myeloid cell surface antigen CD33 isoform 1-precursor [Homo sapiens] MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYY DKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNN CSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIP GTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIIT PRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGK QETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTH PTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNP SKDTSTEYSEVRTQ (11) the transmembrane glycoprotein NMB (SwissProt ID Q14956), SEQ ID NO: 223

>gi|52694752|ref|NP_001005340.1| transmembrane glycoprotein NMB isoform a-precursor [Homo sapiens] MECLYYFLGFLLLAARLPLDAAKRFHDVLGNERPSAYMREHNQLNGWSSD ENDWNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITFAVNL IFPRCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDGENGTGQ SHHNVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSVRVSVNTAN VTLGPQLMEVTVYRRHGRAYVPIAQVKDVYVVTDQIPVFVTMFQKNDRNS SDETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDNTGLFVSTNHTVN HTYVLNGTFSLNLTVKAAAPGPCPPPPPPPRPSKPTPSLATTLKSYDSNT PGPAGDNPLELSRIPDENCQINRYGHFQATITIVEGILEVNIIQMTDVLM PVPWPESSLIDFVVTCQGSIPTEVCTIISDPTCEITQNTVCSPVDVDEMC LLTVRRTFNGSGTYCVNLTLGDDTSLALTSTLISVPDRDPASPLRMANSA LISVGCLAIFVTVISLLVYKKHKEYNPIENSPGNVVRSKGLSVFLNRAKA VFFPGNQEKDPLLKNQEFKGVS (12) the adhesion molecule CD56 (SwissProt ID P13591), SEQ ID NO: 224

>gi|94420689|ref|NP_000606.3| neural cell adhesion molecule 1 isoform 1 [Homo sapiens] MLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVGESKFFLCQVAGDAKDK DISWFSPNGEKLTPNQQRISVVWNDDSSSTLTIYNANIDDAGIYKCVVTG EDGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAVIVCDVVSSLPPTII WKHKGRDVILKKDVRFIVLSNNYLQIRGIKKTDEGTYRCEGRILARGEIN FKDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFPEPTMSWTKD GEQIEQEEDDEKYIFSDDSSQLTIKKVDKNDEAEYICIAENKAGEQDATI HLKVFAKPKITYVENQTAMELEEQVTLTCEASGDPIPSITWRTSTRNISS EEKTLDGHMVVRSHARVSSLTLKSIQYTDAGEYICTASNTIGQDSQSMYL EVQYAPKLQGPVAVYTWEGN QVNITCEVFAYPSATISWFRDGQLLPSSNYSNIKIYNTPSASYLEVTPDS ENDFGNYNCTAVNRIGQESLEFILVQADTPSSPSIDQVEPYSSTAQVQFD EPEATGGVPILKYKAEWRAVGEEVWHSKWYDAKEASMEGIVTIVGLKPET TYAVRLAALNGKGLGEISAASEFKTQPVQGEPSAPKLEGQMGEDGNSIKV NLIKQDDGGSPIRHYLVRYRALSSEWKPEIRLPSGSDHVMLKSLDWNAEY EVYVVAENQQGKSKAAHFVFRTSAQPTAIPANGSPTSGLSTGAIVGILIV IFVLLLVVVDITCYFLNKCGLFMCIAVNLCGKAGPGAKGKDMEEGKAAFS KDESKEPIVEVRTEEERTPNHDGGKHTEPNETTPLTEPEKGPVEAKPECQ ETETKPAPAEVKTVPNDATQTKENESKA (13) the surface molecule CD70 (SwissProt ID P32970), SEQ ID NO: 225

>gi|4507605|ref|NP_001243.1| CD70 antigen [Homo sapiens] MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPL ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHR DGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQG CTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP (14) the surface molecule CD74 (SwissProt ID P04233), SEQ ID NO: 226

>gi|10835071|ref|NP_004346.1| HLA class II histocompatibility antigen gamma chain isoform b [Homo sapiens] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALY TGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKP PKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNAD PLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQ KPTDAPPKESLELEDPSSGLGVTKQDLGPVPM (15) the B-lymphocyte antigen CD19 (SwissProt ID P15391), SEQ ID NO: 227

>gi|296010921|ref|NP_001171569.1| B-lymphocyte antigen CD19 isoform 1-precursor [Homo sapiens] MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQL TWSRESPLKPFLKLSLGLPGLGIHMRPLAIVVLFIFNVSQQMGGFYLCQP GPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSG KLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLS CGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLP RATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAY LIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYG NVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGV GPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGP EDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLAGSQ SYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGG RMGTWSTR (16) the surface protein mucin-1 (SwissProt ID P15941), SEQ ID NO: 228

>gi|65301117|ref|NP_002447.4| mucin-1 isoform 1-precursor [Homo sapiens] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTE KNALSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYK QGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY NLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALA VCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKV SAGNGGSSLSYTNPAVAATSANL (17) the surface protein CD138 (SwissProt ID P18827), SEQ ID NO: 229

>gi|29568086|ref|NP_002988.3| syndecan-1-precursor [Homo sapiens] MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAG ALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPK EGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPAT SHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQL PAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRK EVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQ KPTKQEEFYA (18) the integrin alphaV (Genbank Accession No.: NP_002201.1), SEQ ID NO: 230

>gi|4504763|ref|NP_002201.1|integrin alpha-V isoform 1-precursor [Homo sapiens] MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFG FAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPI EFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQ EREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVL LGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDS YLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGE QMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQ RASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKK GIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYP DLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKV SCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPS HSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAAD TTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVSVDSDQKKIYIG DDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFIGVVRNNEALARLSCAF KTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSS NLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHIFLPIPNWEHKENPETEE DVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPM NCTSDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIH TLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSL KSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILA VLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET (19) the teratocarcinoma-derived growth factor 1 protein TDGF1 (Genbank Accession No.: NP_003203.1), SEQ ID NO: 231

>gi|4507425|ref|NP_003203.1| teratocarcinoma- derived growth factor 1 isoform 1-precursor [Homo sapiens] MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS IWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPS FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY (20) the prostate-specific membrane antigen PSMA (Swiss Prot ID: Q04609), SEQ ID NO: 232

>gi|4758398|ref|NP_004467.1| glutamate carboxypeptidase 2 isoform 1 [Homo sapiens] MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEAT NITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQW KEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPG YENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKI VIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPG GGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYY DAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTN EVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVR SFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKK SPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDY AVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERL QDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKY AGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (21) the tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317), SEQ ID NO: 233

>gi|32967311|ref|NP_004422.2| ephrin type-A receptor 2-precursor [Homo sapiens] MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGK GWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKF TVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEI TVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKK CPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVD GEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPS PEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTP PQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVS DLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTT SLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPD TTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLV LAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQA VLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKA GYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGAL DKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSN LVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDV WSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLM MQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSG SEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVR LPGHQKRIAYSLLGLKDQVNTVGIPI (22) the surface protein SLC44A4 (Genbank Accession No: NP_001171515), SEQ ID NO: 234

>gi|295849282|ref|NP_001171515.1| choline transporter-like protein 4 isoform 2 [Homo sapiens] MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIV VGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNI ISVAENGLQCPTPQTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPAL PGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVL SLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQ LGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLK EASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWAS NISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNL QIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFI RTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFK CCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLD KVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIM TSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLL KILGKKNEAPPDNKKRKK (23) the surface protein BMPR1B (SwissProt: O00238) (24) the transport protein SLC7A5 (SwissProt: Q01650) (25) the epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8) (26) the ovarial carcinoma antigen MUC16 (SwissProt: Q8WXI7) (27) the transport protein SLC34A2 (SwissProt: 095436) (28) the surface protein SEMA5b (SwissProt: Q9P283) (29) the surface protein LYPD1 (SwissProt: Q8N2G4) (30) the endothelin receptor type B EDNRB (SwissProt: P24530) (31) the ring finger protein RNF43 (SwissProt: Q68DV7) (32) the prostate carcinoma-associated protein STEAP2 (SwissProt: Q8NFT2) (33) the cation channel TRPM4 (SwissProt: Q8TD43) (34) the complement receptor CD21 (SwissProt: P20023) (35) the B-cell antigen receptor complex-associated protein CD79b (SwissProt: P40259) (36) the cell adhesion antigen CEACAM6 (SwissProt: P40199) (37) the dipeptidase DPEP1 (SwissProt: P16444) (38) the interleukin receptor IL20Ralpha (SwissProt: Q9UHF4) (39) the proteoglycan BCAN (SwissProt: Q96GW7) (40) the ephrin receptor EPHB2 (SwissProt: P29323) (41) the prostate stem cell-associated protein PSCA (Genbank Accession No: NP_005663.2) (42) the surface protein LHFPL3 (SwissProt: Q86UP9) (43) the receptor protein TNFRSF13C (SwissProt: Q96RJ3) (44) the B-cell antigen receptor complex-associated protein CD79a (SwissProt: P11912) (45) the receptor protein CXCR5 (SwissProt: P32302) (46) the ion channel P2X5 (SwissProt: Q93086) (47) the lymphocyte antigen CD180 (SwissProt: Q99467) (48) the receptor protein FCRL1 (SwissProt: Q96LA6) (49) the receptor protein FCRL5 (SwissProt: Q96RD9) (50) the MHC class II molecule Ia antigen HLA-DOB (Genbank Accession No: NP_002111.1) (51) the T-cell protein VTCN1 (SwissProt: Q7Z7D3) (52) TWEAKR (SEQ ID NO:169 (protein); SEQ ID NO:170 (DNA). (53) the lymphocyte antigen CD37 (Swiss Prot: P11049) (54) the FGF receptor 2; FGFR2 (Gene ID: 2263; official symbol: FGFR2). The FGFR2 receptor occurs in different splice variants (alpha, beta, IIIb, IIIc). All splice variants may act as target molecule. (55) the transmembrane glycoprotein B7H3 (CD276; Gene ID: 80381. (56) the B cell receptor BAFFR (CD268; Gene ID: 115650) (57) the receptor protein ROR 1 (Gene ID: 4919) (58) the surface receptor IL3RA (CD123; Gene ID: 3561) (59) the CXC chemokine receptor CXCR5 (CD185; Gene ID 643) (60) the receptor protein syncytin (Gene ID 30816)

In a preferred subject matter of the invention, the cancer target molecule is selected from the group consisting of the cancer target molecules (1) (60), in particular (1), (6) and (52).

In a further particularly preferred subject matter of the invention, the binder binds to an extracellular cancer target molecule which is selected from the group consisting of the cancer target molecules (1)-(60), in particular (1), (6) and (52).

In a further particularly preferred subject matter of the invention, the binder binds specifically to an extracellular cancer target molecule which is selected from the group consisting of the cancer target molecules (1)-(60), in particular (1), (6) and (52). In a preferred embodiment the binder is, after binding to its extracellular target molecule on the target cell, internalized by the target cell as a result of the binding. This causes the binder/active compound conjugate, which may be an immunoconjugate or an ADC, to be taken up by the target cell. The binder is then processed, preferably intracellularly, with preference lysosomally.

In one embodiment the binder is a binding protein. In a preferred embodiment the binder is an antibody, an aglycosylated antibody, an antigen-binding antibody fragment, a multispecific antibody or an antibody mimetic.

Preferred antibody mimetics are affibodies, adnectins, anticalins, DARPins, avimers, or nobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies.

In a preferred embodiment the binder is an antibody or an antigen-binding antibody fragment, more preferably an isolated antibody or an isolated antigen-binding antibody fragment.

Preferred antigen-binding antibody fragments are Fab, Fab′, F(ab′)2 and Fv fragments, diabodies, DAbs, linear antibodies and scFv. Particularly preferred are Fab, diabodies and scFv.

In a particularly preferred embodiment the binder is an antibody. Particularly preferred are monoclonal antibodies or antigen-binding antibody fragments thereof. Further particularly preferred are human, humanized or chimeric antibodies or antigen-binding antibody fragments thereof.

Antibodies or antigen-binding antibody fragments which bind cancer target molecules may be prepared by a person of ordinary skill in the art using known processes, such as, for example, chemical synthesis or recombinant expression. Binders for cancer target molecules may be acquired commercially or may be prepared by a person of ordinary skill in the art using known processes, such as, for example, chemical synthesis or recombinant expression. Further processes for preparing antibodies or antigen-binding antibody fragments are described in WO 2007/070538 (see page 22 “Antibodies”). The person skilled in the art knows how processes such as phage display libraries (e.g. Morphosys HuCAL Gold) can be compiled and used for discovering antibodies or antigen-binding antibody fragments (see WO 2007/070538, page 24 ﬀ and AK Example 1 on page 70, AK Example 2 on page 72). Further processes for preparing antibodies that use DNA libraries from B cells are described for example on page 26 (WO 2007/070538). Processes for humanizing antibodies are described on page 30-32 of WO2007070538 and in detail in Queen, et al., Pros. Natl. Acad. Sci. USA 86:10029-10033, 1989 or in WO 90/0786. Furthermore, processes for the recombinant expression of proteins in general and of antibodies in particular are known to the person skilled in the art (see, for example, in Berger and Kimmel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.], Current Protocols, Green Publishing Associates, Inc./John Wiley & Sons, Inc.); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988, Paul [Ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al., (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)). The person skilled in the art knows the corresponding vectors, promoters and signal peptides which are necessary for the expression of a protein/antibody. Commonplace processes are also described in WO 2007/070538 on pages 41-45. Processes for preparing an IgG1 antibody are described for example in WO 2007/070538 in Example 6 on page 74 ﬀ. Processes which allow the determination of the internalization of an antibody after binding to its antigen are known to the skilled person and are described for example in WO 2007/070538 on page 80. The person skilled in the art is able to use the processes described in WO 2007/070538 that have been used for preparing carboanhydrase IX (Mn) antibodies in analogy for the preparation of antibodies with different target molecule specificity.

Anti-EGFR Antibodies

Examples of antibodies which bind the cancer target molecules EGFR are cetuximab (INN number 7906), panitumumab (INN number 8499) and nimotuzumab (INN number 8545). Cetuximab (Drug Bank Accession Number DB00002) is a chimeric anti-EGFR1 antibody which is produced in SP2/0 mouse myeloma cells and is sold by ImClone Systems Inc/Merck KgaA/Bristol-Myers Squibb Co. Cetuximab is indicated for the treatment of metastasizing, EGFR expressing, colorectal carcinoma with wild type K-Ras gene. It has an affinity of 10⁻¹⁰ M.

Sequence:

Cetuximab Light Chain (kappa), SEQ ID NO: 235: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Cetuximab Heavy Chain, SEQ ID NO: 236: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALT YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Panitumumab (INN number 8499) (Drug Bank Accession Number DB01269) is a recombinant monoclonal human IgG2 antibody which binds specifically to the human EGF receptor 1 and is sold by Abgenix/Amgen. Panitumumab originates from the immunization of transgenic mice (XenoMouse). These mice are capable of producing human immunoglobulin (light and heavy chains). A specific B-cell clone was selected which produces antibodies against EGFR, and this clone was immortalized with CHO cells (Chinese hamster ovary cells). These cells are now used for the production of a 100% human antibody. Panitumumab is indicated for the treatment of EGFR-expressing, metastasizing colorectal carcinoma, which is resistant to chemotherapeutic treatment with fluoropyrimidine, oxaliplatin and irinotecan. It has an affinity of 10⁻¹¹M.

Sequence:

Panitumumab Light Chain (kappa), SEQ ID NO: 237: DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY DASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Panitumumab Heavy Chain, SEQ ID NO: 238: QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEW IGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCV RDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFG TQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG

Nimotuzumab (INN number 8545) (EP 00586002, EP 00712863) is a humanized monoclonal IgG1 antibody which binds specifically to the human EGF receptor 1 and is sold by YM BioScienecs Inc. (Mississauga Canada). It is produced in non-secreting NSO cells (mammalian cell line). Nimotuzumab is approved for the treatment of head-and-neck tumours, highly malignant astrocytoma and glioblastoma multiforms (not in EU and US) and pancreatic carcinoma (Orphan drug, EMA). It has an affinity of 10⁻⁸ M.

Nimotuzumab Light Chain, SEQ ID NO: 239: DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKA PKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQY SHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC Nimotuzumab Heavy Chain, SEQ ID NO: 240: QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWI GGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFC TRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK

Further embodiments of EGFR antibodies are as follows:

-   -   Zalutumumab/2F8/HuMax-EGFr, from Genmab A/S (WO 02/100348, WO         2004/056847, INN number 8605)     -   Necitumumab/11F8, ImClone/IMC-11F8, from ImClone Systems Inc.         [Eli Lilly & Co] (WO 2005/090407 (EP 01735348-A1, US         2007/0264253-A1, U.S. Pat. No. 7,598,350, WO 2005/090407-A1),         INN number 9083)     -   Matuzumab/anti-EGFR MAb, Merck KGaA/anti-EGFR MAb, Takeda/EMD         72000/EMD-6200/EMD-72000 and EMD-55900/MAb 425/monoclonal         antibody 425, from Merck KGaA/Takeda (WO 92/15683, INN number         8103 (Matuzumab))     -   RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/R04858696/R05083945,         from Glycart Biotechnology AG (Roche Holding AG) (WO         2010/112413-A1, WO 2010/115554)     -   GT-MAB 5.2-GEX/CetuGEX, from Glycotope GmbH (WO 2008/028686-A2         (EP 01900750-A1, EP 01911766-A1, EP 02073842-A2, US         2010/0028947-A1)     -   ISU-101, from Isu Abxis Inc (ISU Chemical Co Ltd)/Scancell (WO         2008/004834-A1)     -   ABT-806/mAb-806/ch-806/anti-EGFR monoclonal antibody 806, from         Ludwig Institute for Cancer Research/Abbott/Life Science         Pharmaceuticals (WO 02/092771, WO 2005/081854 and WO         2009/023265)     -   SYM-004 (consists of two chimeric IgG1 antibodies (992 and         1024)), from Symphogen A/S (WO 2010/022736-A2)     -   MR1-1/MR1-1KDEL, from IVAX Corp (Teva Pharmaceutical Industries         Ltd) (Duke University), (patent: WO2001/062931-A2)     -   Antibody against the deletion mutant, EGFRvIII, from         Amgen/Abgenix (WO 2005/010151, U.S. Pat. No. 7,628,986)     -   SC-100, from Scancell Ltd (WO 01/088138-A1)     -   MDX-447/EMD 82633/BAB-447/H447/MAb, EGFR, Medarex/Merck KgaA,         from Bristol-Myers Squibb (US)/Merck KGaA (DE)/Takeda (JP), (WO         91/05871, WO 92/15683)     -   anti-EGFR-Mab, from Xencor (WO 2005/056606)     -   DXL-1218/anti-EGFR monoclonal antibody (cancer), InNexus, from         InNexus Biotechnology Inc, Pharmaprojects PH048638

In a preferred embodiment, the anti-EGFR antibodies are selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab, RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218.

In a particularly preferred embodiment the anti-EGFR antibodies are selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab and matuzumab.

The person skilled in the art knows of processes which can be used to prepare further antibodies, from the CDR regions of the abovementioned antibodies by means of sequence variations, these further antibodies having a similar or better affinity and/or specificity for the target molecule. Furthermore the person skilled in the art knows of processes which can be used to prepare further antibodies, from the CDR regions of the abovementioned antibodies by means of sequence variations, these further antibodies are aglycosylated and/or engineered to contain one or more acceptor glutamine residues for transglutaminase (TGase) catalyzed reactions.

In a further embodiment, the anti-EGFR antibodies or antigen-binding antibody fragments are selected from the group consisting of

antibodies or antigen-binding antibody fragments comprising the three CDR regions of the light chain and the three CDR regions of the heavy chain of one of the following antibodies: cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab, RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and DXL-1218.

In a further embodiment, the anti-EGFR antibodies or antigen-binding antibody fragments are selected from the group consisting of

antibodies or antigen-binding antibody fragments comprising three CDR regions of the light chain and the three CDR regions of the heavy chain of one of the following antibodies: cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention. Anti-Carboanhydrase IX Antibodies

Examples of antibodies which bind the cancer target molecule carbonahydrase IX are described in WO 2007/070538-A2 (e.g. Claims 1-16).

In a preferred embodiment the anti-carboanhydrase IX antibodies or antigen-binding antibody fragments are selected from the group consisting of anti-carboanhydrase IX antibodies or antigen-binding antibody fragments 3ee9 (claim 4 (a) in WO 2007/070538-A2), 3ef2 (claim 4 (b) in WO2007/070538-A2), 1e4 (claim 4 (c) in WO 2007/070538-A2), 3a4 (claim 4 (d) in WO 2007/070538-A2), 3ab4 (claim 4 (e) in WO 2007/070538-A2), 3ah10 (claim 4 (f) in WO 2007/070538-A2), 3bb2 (claim 4 (g) in WO 2007/070538-A2), 1aa1 (claim 4 (h) in WO 2007/070538-A2), 5a6 (claim 4 (i) in WO 2007/070538-A2) and 5aa3 (claim 4 (j) in WO 2007/070538-A2).

Anti-C4.4a Antibodies:

According to the invention, use may be made of C4.4a antibodies.

Examples of C4.4a antibodies and antigen-binding fragments are described in WO 2012/143499 A2. By reference, all antibodies of WO 2012/143499 A2 are hereby incorporated into the description of the present invention, and they can be used in the present invention. The sequences of the antibodies are given in Table 1 of WO 2012/143499 A2, where each row shows the respective CDR amino acid sequences of the variable light chain or the variable heavy chain of the antibody listed in column 1.

In one embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments thereof are, after binding to a cell expressing C4.4a, internalized by the cell.

In a further embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments comprise at least one, two or three CDR amino acid sequences of an antibody listed in Table 1 of WO 2012/143499 A2 or Table 2 of WO 2012/143499 A2. Preferred embodiments of such antibodies are likewise listed in WO 2012/143499 A2 and incorporated herein by reference.

Anti-HER2 Antibodies:

An example of an antibody binding to the cancer target molecule Her2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody used inter alia for the treatment of breast cancer.

Further examples of antibodies binding to HER2 are, in addition to trastuzumab (INN 7637, CAS No.: RN: 180288-69-1) and Pertuzumab (CAS No.: 380610-27-5), the antibodies disclosed in WO 2009/123894-A2, WO 200/8140603-A2 or in WO 2011/044368-A2. An example of an anti-HER2 conjugate is trastuzumab-emtansine (INN-No. 9295). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD20 Antibodies:

An example of an antibody binding to the cancer target molecule CD20 is rituximab (Genentech). Rituximab (CAS Number: 174722-31-7) is a chimeric antibody used for the treatment of non-Hodgkin lymphoma. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD52 Antibodies:

An example of an antibody binding to the cancer target molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS Number: 216503-57-0) is a humanized antibody used for the treatment of chronic lymphocytic leukaemia. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-Mesothelin Antibodies:

Examples of anti-mesothelin antibodies are described, for example, in WO 2009/068204. By reference, all antibodies described in WO 2009/068204 are hereby incorporated into the present description, such that these antibodies can be used in the context of the invention disclosed herein.

The anti-mesothelin antibodies used in accordance with the invention are also notable preferably for an invariant binding to mesothelin. Invariant binding is characterized, for example, in that the antibody used in accordance with the invention binds to an epitope of mesothelin which cannot be masked by a further extracellular protein. Such a further extracellular protein is, for example, the protein ovarian cancer antigen 125 (CA125). Antibodies which are used with preference are characterized in that their binding to mesothelin is not blocked by CA125.

Anti-CD30 Antibodies

Examples of antibodies which bind the cancer target molecule CD30 and can be used for the treatment of cancer, for example Hodgkin lymphoma, are brentuximab, iratumumab and antibodies disclosed in WO 2008/092117, WO 2008/036688 or WO 2006/089232. An example of an anti-CD30 conjugate is brentuximab vedotin (INN No. 9144). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD22 Antibodies

Examples of antibodies which bind the cancer target molecule CD22 and can be used for the treatment of cancer, for example lymphoma, are inotuzumab and epratuzumab. Examples of anti-CD22 conjugates are inotuzumab ozagamycin (INN No. 8574) or anti-CD22-MMAE and anti-CD22-MC-MMAE (CAS RN: 139504-50-0 and 474645-27-7, respectively). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD33 Antibodies

Examples of antibodies which bind the cancer target molecule CD33 and can be used for the treatment of cancer, for example leukaemia, are gemtuzumab and lintuzumab (INN 7580). An example of an anti-CD33 conjugate is gemtuzumab-ozagamycin. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-NMB Antibodies

An example of an antibody which binds the cancer target molecule NMB and can be used for the treatment of cancer, for example melanoma or breast cancer, is glembatumumab (INN 9199). An example of an anti-NMB conjugate is glembatumumab vedotin (CAS RN: 474645-27-7). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD56 Antibodies

An example of an antibody which binds the cancer target molecule CD56 and can be used for the treatment of cancer, for example multiple myeloma, small-cell lung carcinoma, MCC or ovarial carcinoma is lorvotuzumab. An example of an anti-CD56 conjugate is lorvotuzumab mertansine (CAS RN: 139504-50-0). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD70 Antibodies

Examples of antibodies which bind the cancer target molecule CD70 and can be used for the treatment of cancer, for example non-Hodgkin lymphoma or renal cell cancer, are disclosed in WO 2007/038637-A2 and WO 2008/070593-A2. An example of an anti-CD70 conjugate is SGN-75 (CD70 MMAF). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD74 Antibodies

An example of an antibody which binds the cancer target molecule CD74 and can be used for the treatment of cancer, for example multiple myeloma, is milatuzumab. An example of an anti-CD74 conjugate is milatuzumab-doxorubicin (CAS RN: 23214-92-8). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD19 Antibodies

An example of an antibody which binds the cancer target molecule CD19 and can be used for the treatment of cancer, for example non-Hodgkin lymphoma, is disclosed in WO 2008/031056-A2. Further antibodies and examples of an anti-CD19 conjugate (SAR3419) are disclosed in WO 2008/047242-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-Mucin Antibodies

Examples of antibodies which bind the cancer target molecule mucin-1 and can be used for the treatment of cancer, for example non-Hodgkin lymphoma, are clivatuzumab and the antibodies disclosed in WO 2003/106495-A2, WO 2008/028686-A2. Examples of anti-mucin conjugates are disclosed in WO 2005/009369-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-CD138 Antibodies

Examples of antibodies which bind the cancer target molecule CD138 and conjugates thereof, which can be used for the treatment of cancer, for example multiple myeloma, are disclosed in WO 2009/080829-A1, WO 2009/080830-A1. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-Integrin-alphaV Antibodies

Examples of antibodies which bind the cancer target molecule integrin alphaV and can be used for the treatment of cancer, for example melanoma, sarcoma or carcinoma, are intetumumab (CAS RN: 725735-28-4), abciximab (CAS RN: 143653-53-6), etaracizumab (CAS RN: 892553-42-3) and the antibodies disclosed in U.S. Pat. No. 7,465,449, EP 719859-A1, WO 2002/012501-A1 and WO2006/062779-A2. Examples of anti-integrin AlphaV conjugates are intetumumab-DM4 and other ADCs disclosed in WO 2007/024536-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-TDGF1 Antibodies

Examples of antibodies which bind the cancer target molecule TDGF1 and can be used for the treatment of cancer are the antibodies disclosed in WO 02/077033-A1, U.S. Pat. No. 7,318,924, WO 2003/083041-A2 and WO 2002/088170-A2. Examples of anti-TDGF1 conjugates are disclosed in WO 2002/088170-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-PSMA Antibodies

Examples of antibodies which bind the cancer target molecule PSMA and can be used for the treatment of cancer, for example prostate carcinoma, are the antibodies disclosed in WO 97/35616-A1, WO 99/47554-A1, WO 01/009192-A1 and WO2003/034903. Examples of anti-PSMA conjugates are disclosed in WO 2009/026274-A1 and WO 2007/002222. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-EPHA2 Antibodies

Examples of antibodies which bind the cancer target molecule EPHA2 and can be used for preparing a conjugate and for the treatment of cancer are disclosed in WO 2004/091375-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-SLC44A4 Antibodies

Examples of antibodies which bind the cancer target molecule SLC44A4 and can be used for preparing a conjugate and for the treatment of cancer, for example pancreas or prostate carcinoma, are disclosed in WO2009/033094-A2 and US2009/0175796-A1. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-HLA-DOB Antibodies

An example of an antibody binding to the cancer target molecule HLA-DOB is the antibody Lym-1 (CAS RN: 301344-99-0) which can be used for the treatment of cancer, for example non-Hodgkin lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, for example, in WO 2005/081711-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-VTCN1 Antibodies

Examples of antibodies which bind the cancer target molecule VTCN1 and can be used for preparing a conjugate and for the treatment of cancer, for example ovarial carcinoma, pancreas, lung or breast cancer, are disclosed in WO 2006/074418-A2. By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention.

Anti-FGFR2 Antibodies

According to the invention, use may be made of anti-FGFR2 antibodies.

Examples of anti-FGFR2 antibodies and antigen-binding fragments are described in WO2013076186. By reference, all antibodies of WO2013076186 are hereby incorporated into the description of the present invention, and they can be used in the present invention. The sequences of the antibodies are shown in Table 9 and Table 10 of WO2013076186. Preference is given to antibodies, antigen-binding fragments and variants of the antibodies derived from the antibodies referred to as M048-D01 and M047-D08. Preferred anti-FGFR2 bind to the various splice variants known of FGFR2.

In one embodiment, the anti-FGFR2 antibodies or antigen-binding antibody fragments thereof are, after binding to a cell expressing FGFR2, internalized by the cell.

In a further embodiment, the anti-FGFR2 antibodies or antigen-binding antibody fragments comprise at least one, two or three CDR amino acid sequences of an antibody listed in Table 9 or Table 10 of WO2013076186. Preferred embodiments of such antibodies are likewise listed in WO2013076186 and incorporated herein by reference.

Anti-TWEAKR Antibodies

In a preferred embodiment, when an anti-TWEAKR antibody or an antigen-binding fragment thereof is used in the processes according to the present invention, this antibody or fragment is selected from those described below. In addition, antibodies which bind to TWEAKR are known to the person skilled in the art, see, for example, WO 2015/189143 (A1), WO 2014/198817 (A1), WO2009/020933(A2) or WO2009140177 (A2). In addition, aglycosylated variants of the described anti-TWEAKR antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid are used in the processes according to the present invention. Furthermore variants of these antibodies being engineered to contain one or more acceptor glutamine residues for transglutaminase (TGase) catalyzed reactions.

The invention relates in particular to conjugates with antibodies or antigen-binding antibody fragments thereof or variants thereof which lead to strong activation of the TWEAKR (SEQ ID NO:169 (protein); SEQ ID NO:170 (DNA)), resulting in a strong induction of apoptosis in various cancer cells overexpressing TWEAKR.

The agonistic activity of TWEAKR with regard to the induction of apoptosis and inhibition of the proliferation of the anti-TWEAKR antibodies already described (e.g. PDL-192) is limited and does not reach the efficacy of the endogenous ligand TWEAK. This lack of agonistic activity is not based on reduced affinity, since these antibodies bind at the TWEAKR with affinities which, compared to the endogenous ligand TWEAK, are in a similar range (Michaelson J S et al, MAbs. 2011 July-August; 3(4):362-75; Culp P A et al, Clin Cancer Res. 2010 Jan. 15; 16(2):497-508), and even antibodies having a higher binding affinity do not necessarily display a more effective signalling activity (Culp P A, et al, Clin Cancer Res. 2010 Jan. 15; 16(2):497-508). In addition, it has been shown that the antitumour activity of the antibodies already described depends on the Fc effector function, and it was shown that ADCC plays an important role for the in-vivo efficacy in mouse models.

Generation of the Anti-TWEAKR Antibodies

A complete human antibody phage library (Hoet R M et al, Nat Biotechnol 2005; 23(3):344-8) was employed to isolate TWEAKR-specific human monoclonal antibodies of the present invention by protein panning (Hoogenboom H. R., Nat Biotechnol 2005; 23(3):1105-16) using dimeric Fc-fused extracellular domains of human and mouse TWEAKR as immobilized target. 11 different Fab phages were identified, and the corresponding antibodies were cloned into a mammalian EgG expression vector which provides the CH2-CH3 domains missing in the soluble FAb. Following identification of preferred antibodies, these were expressed as full-length IgGs. Aglycosylated variants of the described antibodies were generated by introducing the mutation N297A or N297Q in the heavy chain of the respective antibody. The constructs were expressed, for example, transiently in mammalian cells as described by Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007 (see AK-Example 1). The antibodies were purified by protein-A chromatography and characterized further by their binding affinity to soluble monomeric TWEAKR using ELISA and BIAcore analysis, as described in AK-Example 2. To determine the cell binding characteristics of the anti-TWEAKR antibodies, binding was tested by flow cytometry on a number of cell lines (HT29, HS68, HS578). NFκB reporter gene assays were carried out to examine the agonistic activity of all 11 antibodies identified (human IgG1). The antibody having the highest in vitro activity (TPP-883) was selected for further activity and affinity maturation (see AK-Example 1 for details). A single substitution variant having improved agonistic activity was detected: G102T of CDR-H3. In the end, 7 variants were selected based on increased affinity compared to the best single substitution variant G102T. The corresponding DNA thereof was cloned into a mammalian IgG expression vector and examined for functional activity in the NF-kappaB reporter gene assay mentioned above. Finally, the sequences obtained were compared with human germ line sequences, and deviations without any significant effect on the affinity and the efficacy were adapted. The following antibodies were obtained by antibody library screening and by affinity and/or activity maturation. “TPP-2090”, “TPP-2149”, “TPP-2093”, “TPP-2148”, “TPP-2084”, “TPP-2077”, “TPP-1538”, “TPP-883”, “TPP-1854”, “TPP-1853”, “TPP-1857” and “TPP-1858”.

Antibodies of the invention can furthermore be obtained by methods known in the art such as antibody phage display screening (see, for example, Hoet R M et al., Nat Biotechnol 2005; 23(3):344-8), the well-established hybridoma technology (see, for example, Köhler and Milstein Nature. 1975 Aug. 7; 256(5517):495-7) or immunization of mice, inter alia immunization of hMAb mice (e.g. VelocImmune Mouse®).

Particular Embodiments of Anti-TWEAKR Antibodies

One embodiment of the invention is the provision of antibodies or antigen-binding antibody fragments thereof or variants thereof showing strong induction of caspase 3/7 in one or more TWEAKR-expressing cell lines (see also WO 2015/189143 A1 and WO 2014/198817 A1). In a preferred embodiment, the one or more TWEAKR-expressing cell line(s) is/are present in the group consisting of WiDr, A253, NCI-H322, HT29 and 786-O. “Induction of caspase 3/7” can be measured by customary methods known in the art, including those described herein. In one embodiment, the “induction of caspase 3/7” is determined in accordance with the present invention using the activity determination with capase 3/7 solution (Promega, #G8093) and reading the luminescence on a VICTOR V (Perkin Elmer). At the end of the incubation time, the caspase 3/7 activity was determined and the induction factor of caspase 3/7 was determined in comparison to untreated cells. An antibody is said to show “strong induction” of caspase 3/7 when the induction factor is greater than 1.2, preferably greater than 1.5, even more preferably greater than 1.8, even more preferably greater than 2.1, even more preferably greater than 2.5. What is provided are anti-TWEAKR antibodies leading to stronger induction of caspase 3/7 in HT29 cells compared to agonistic antibodies already described [e.g. PDL-192(TPP-1104), P4A8(TPP-1324), 136.1(TPP-2194)] and also compared to 300 ng/ml recombinant human TWEAK. This strong activity of inducing caspase 3/7 in cancer cells was also observed in WiDr, A253, NIC-H322 and 786-O cells where in most experiments the antibodies of the invention examined induced higher factors of change compared to the reference antibodies [PDL-192(TPP-1104), P4A8(TPP-1324)] and to 300 ng/ml TWEAK. Some antibodies of the invention bind to the TWEAKR only with morate affinity (>10 nM) which is clearly less than the affinity of the endogenous ligand TWEAK, and also less compared to other known agonistic antibodies. This property offers further possible advantages such as, for example, potentially deeper penetration into the tumour.

In this regard, one embodiment of the invention is the provision of antibodies or antigen-binding antibody fragments thereof binding specifically to a TWEAKR at a novel epitope characterized by selective binding to aspartate (D) at position 47 (D47) of TWEAKR (SEQ ID NO:169; see also FIG. 1 ). The dependencies identified for certain TWEAKR amino acids for antibody interaction correlate with the agonistic activity determined for these antibodies. The native ligand TWEAK shows an effective activation of the TWEAKR and binds depending on leucine 46 in the cysteine-rich domain of TWEAKR (Pellegrini et al, FEBS 280:1818-1829). P4A8 displays a very low agonistic activity and interacts at least partially with domains outside of the cysteine-rich domain of TWEAKR. PDL-192 displays a morate agonistic activity and binds depending on R56 to the cysteine-rich domain, but opposite the TWEAK ligand site. Antibodies of the present invention (e.g. TPP-2090) bind depending on D47, and TWEAK binds depending on L46. Thus, TWEAK binds to a similar but different binding site FIG. 7 ). Accordingly, the antibodies of the present invention displaying strong agonistic activity bind to a novel epitope (D47-dependent) for antibodies associated with very high agonistic activity.

The amino acid at position 47 (D47) of TWEAKR (SEQ ID NO:169) is considered to be critical for binding of the antibodies according to the invention, which means that the antibody binds specifically to the D at position 47 (D47) of TWEAKR (SEQ ID NO:169) when the antibody loses more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, alternatively more than 70%, alternatively more than 80%, alternatively more than 90%, alternatively 100% of its ELISA signal by modification of this residue into alanine, as described in AK-Example 2 and FIG. 6 . Alternatively, an antibody binds specifically to the D at position 47 (D47) of TWEAKR (SEQ ID NO:169) when the antibody loses more than 20%, alternatively more than 30%, alternatively more than 40%, alternatively more than 50%, alternatively more than 60%, alternatively more than 70%, alternatively more than 80%, alternatively more than 90%, alternatively 100% of its ELISA signal for TPP-2614 compared to TPP-2203. Preferably, an antibody binds specifically to the D at position 47 (D47) of TWEAKR (SEQ ID NO:169) when the antibody loses more than 80% of its ELISA signal for TPP-2614 compared to TPP-2203.

In the present application, reference is made to the following preferred antibodies of the invention, as shown in the table below: “TPP-2090”, “TPP-2149”, “TPP-2093”, “TPP-2148”, “TPP-2084”, “TPP-2077”, “TPP-1538”, “TPP-883”, “TPP-1854”, “TPP-1853”, “TPP-1857”, “TPP-1858”.

TABLE Protein sequences of the antibodies: SEQ ID SEQ ID NO: IgG1 NO: IgG1 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: light chain heavy chain L-CDR1 L-CDR2 L-CDR3 H-CDR1 Anti-TWEAKR antibodies according to the invention TPP-2090 1 2 3 4 5 6 TPP-2149 11 12 13 14 15 16 TPP-2093 2 22 23 24 25 26 TPP-2148 31 32 33 34 35 36 TPP-2084 41 42 43 44 45 46 TPP-2077 51 52 53 54 55 56 TPP-1538 61 62 63 64 65 66 TPP-883 71 72 73 74 75 76 TPP-1854 81 82 00 84 85 86 TPP-1853 91 92 93 94 95 96 TPP-1857 101 102 103 104 105 106 TPP-1858 111 112 113 114 115 116 TPP-2658 1 241 3 4 5 6 TPP-5442 1 242 3 4 5 6 TPP-8825 1 243 3 4 5 6 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: H-CDR2 H-CDR3 VL protein VH protein Anti-TWEAKR antibodies according to the invention TPP-2090 7 8 9 10 TPP-2149 17 18 19 20 TPP-2093 27 28 29 30 TPP-2148 37 38 39 40 TPP-2084 47 48 49 50 TPP-2077 57 58 59 60 TPP-1538 67 68 69 70 TPP-883 77 78 79 80 TPP-1854 87 88 89 90 TPP-1853 97 98 99 100 TPP-1857 107 108 109 110 TPP-1858 117 118 119 120 TPP-2658 7 8 9 10 TPP-5442 7 8 9 10 TPP-8825 7 8 9 10 SEQ ID SEQ ID NO: IgG1 NO: IgG1 light chain heavy chain Comparative antibodies: P3G5(TPP-2195) 121 122 136.1(TPP-2194) 123 124 P4A8(TPP-1324) 125 126 PDL-192(TPP-1104) 127 128 18.3.3(TPP-2193) 129 130 P2D3(TPP-2196) 131 132

-   -   TPP-2090 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 2 and a region of the light         chain corresponding to SEQ ID NO: 1.     -   TPP-2658 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 241 and a region of the light         chain corresponding to SEQ ID NO: 1.     -   TPP-5442 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 242 and a region of the light         chain corresponding to SEQ ID NO: 1.     -   TPP-8825 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 243 and a region of the light         chain corresponding to SEQ ID NO: 1.     -   TPP-2149 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 12 and a region of the light         chain corresponding to SEQ ID NO: 11.     -   TPP-2093 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 22 and a region of the light         chain corresponding to SEQ ID NO: 21.     -   TPP-2148 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 32 and a region of the light         chain corresponding to SEQ ID NO: 31.     -   TPP-2084 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 42 and a region of the light         chain corresponding to SEQ ID NO: 41.     -   TPP-2077 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 52 and a region of the light         chain corresponding to SEQ ID NO: 51.     -   TPP-1538 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 62 and a region of the light         chain corresponding to SEQ ID NO: 61.     -   TPP-883 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 72 and a region of the light         chain corresponding to SEQ ID NO: 71.     -   TPP-1854 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 82 and a region of the light         chain corresponding to SEQ ID NO: 81.     -   TPP-1853 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 92 and a region of the light         chain corresponding to SEQ ID NO: 91.     -   TPP-1857 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 102 and a region of the light         chain corresponding to SEQ ID NO: 101.     -   TPP-1858 is: an antibody which comprises a region of the heavy         chain corresponding to SEQ ID NO: 112 and a region of the light         chain corresponding to SEQ ID NO: 111.     -   TPP-2090 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 10 and a variable         region of the light chain corresponding to SEQ ID NO: 9.     -   TPP-2149 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 20 and a variable         region of the light chain corresponding to SEQ ID NO: 19.     -   TPP-2093 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 30 and a variable         region of the light chain corresponding to SEQ ID NO: 29.     -   TPP-2148 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 40 and a variable         region of the light chain corresponding to SEQ ID NO: 39.     -   TPP-2084 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 50 and a variable         region of the light chain corresponding to SEQ ID NO: 49.     -   TPP-2077 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 60 and a variable         region of the light chain corresponding to SEQ ID NO: 59.     -   TPP-1538 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 70 and a variable         region of the light chain corresponding to SEQ ID NO: 69.     -   TPP-883 is: an antibody which comprises a variable region of the         heavy chain corresponding to SEQ ID NO: 80 and a variable region         of the light chain corresponding to SEQ ID NO: 79.     -   TPP-1854 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 90 and a variable         region of the light chain corresponding to SEQ ID NO: 89.     -   TPP-1853 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 100 and a variable         region of the light chain corresponding to SEQ ID NO: 99.     -   TPP-1857 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 110 and a variable         region of the light chain corresponding to SEQ ID NO: 109.     -   TPP-1858 is: an antibody which comprises a variable region of         the heavy chain corresponding to SEQ ID NO: 120 and a variable         region of the light chain corresponding to SEQ ID NO: 119.

TABLE DNA sequences of antibodies according to the invention SEQ ID NO: SEQ ID NO: Antibody IgG1 light chain IgG1 heavy chain Antibodies according to the invention: TPP-2090 177 178 TPP-2149 179 180 TPP-2093 181 182 TPP-2148 183 184 TPP-2084 185 186 TPP-2077 187 188 TPP-1538 189 190 TPP-883 191 192 TPP-1854 193 194 TPP-1853 195 196 TPP-1857 197 198 TPP-1858 199 200 Comparative antibodies: P3G5(TPP-2195) 201 202 136.1(TPP-2194) 203 204 P4A8(TPP-1324) 205 206 PDL-192(TPP-1104) 207 208 18.3.3(TPP-2193) 209 210 P2D3(TPP-2196) 211 212

Preferred embodiments of the anti-TWEAKR antibody are those below:

-   -   1. An anti-TWEAKR antibody, an aglycosylated variant, or an         antigen-binding fragment thereof which binds specifically to the         D at position 47 (D47) of the TWEAKR (SEQ ID NO:169).     -   2. The antibody or an antigen-binding fragment thereof according         to embodiment 1 where the antibody is an agonistic antibody.     -   3. The antibody or an antigen-binding fragment thereof according         to embodiment 1 or 2 which comprises:         -   a variable heavy chain comprising:         -   (a) a CDR1 of the heavy chain encoded by an amino acid             sequence comprising the formula PYPMX (SEQ ID NO: 171),             where X is I or M;         -   (b) a CDR2 of the heavy chain encoded by an amino acid             sequence comprising the formula YISPSGGXTHYADSVKG (SEQ ID             NO: 172), where X is S or K; and         -   (c) a CDR3 of the heavy chain encoded by an amino acid             sequence comprising the formula GGDTYFDYFDY (SEQ ID NO:             173);             -   and a variable light chain comprising:         -   (a) a CDR1 of the light chain encoded by an amino acid             sequence comprising the formula RASQSISXYLN (SEQ ID NO:             174), where X is G or S;         -   (b) a CDR2 of the light chain encoded by an amino acid             sequence comprising the formula XASSLQS (SEQ ID NO: 175),             where X is Q, A or N; and         -   (c) a CDR3 of the light chain encoded by an amino acid             sequence comprising the formula QQSYXXPXIT (SEQ ID NO: 176),             where X at position 5 is T or S, X at position 6 is T or S             and X at position 8 is G or F.     -   4. The antibody or an antigen-binding fragment thereof according         to any of the preceding embodiments, comprising:         -   a. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 6, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 7 and the variable CDR3 sequence of the heavy chain,             as shown in SEQ ID NO: 8, and also a variable light chain             comprising the variable CDR1 sequence of the light chain             shown in SEQ ID NO: 3, the variable CDR2 sequence of the             light chain shown in SEQ ID NO: 4 and the variable CDR3             sequence of the light chain shown in SEQ ID NO: 5 or         -   b. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 16, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 17, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:18, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 13, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 14 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:15 or         -   c. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 26, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 27, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:28, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 23, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 24 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:25 or         -   d. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 36, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 37, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:38, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 33, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 34 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:35 or         -   e. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 46, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 47, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:48, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 43, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 44 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:45 or         -   f. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 56, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 57, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:58, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 53, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 54 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:55 or         -   g. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 66, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 67, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:68, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 63, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 64 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:65 or         -   h. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 76, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 77, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:78, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 73, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 74 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:75 or         -   i. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 86, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 87, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:88, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 83, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 84 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:85 or         -   j. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 96, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 97, the variable CDR3 sequence of the heavy chain, as             shown in SEQ ID NO:98, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 93, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 94 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:95 or         -   k. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 106, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 107, the variable CDR3 sequence of the heavy chain,             as shown in SEQ ID NO:108, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 103, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 104 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:105 or         -   l. a variable heavy chain comprising the variable CDR1             sequence of the heavy chain, as shown in SEQ ID NO: 116, the             variable CDR2 sequence of the heavy chain, as shown in SEQ             ID NO: 117, the variable CDR3 sequence of the heavy chain,             as shown in SEQ ID NO:118, and also             -   a variable light chain comprising the variable CDR1                 sequence of the light chain shown in SEQ ID NO: 113, the                 variable CDR2 sequence of the light chain shown in SEQ                 ID NO: 114 and the variable CDR3 sequence of the light                 chain shown in SEQ ID NO:115.     -   5. The antibody or the antigen-binding fragment thereof         according to any of the preceding embodiments, comprising:         -   a. a variable sequence of the heavy chain, as shown in SEQ             ID NO:10, and also a variable sequence of the light chain,             as shown in SEQ ID NO:9, or         -   b. a variable sequence of the heavy chain, as shown in SEQ             ID NO:20, and also a variable sequence of the light chain,             as shown in SEQ ID NO:19, or         -   c. a variable sequence of the heavy chain, as shown in SEQ             ID NO:30, and also a variable sequence of the light chain,             as shown in SEQ ID NO:29, or         -   d. a variable sequence of the heavy chain, as shown in SEQ             ID NO:40, and also a variable sequence of the light chain,             as shown in SEQ ID NO:39, or         -   e. a variable sequence of the heavy chain, as shown in SEQ             ID NO:50, and also a variable sequence of the light chain,             as shown in SEQ ID NO:49, or         -   f. a variable sequence of the heavy chain, as shown in SEQ             ID NO:60, and also a variable sequence of the light chain,             as shown in SEQ ID NO:59, or         -   g. a variable sequence of the heavy chain, as shown in SEQ             ID NO:70, and also a variable sequence of the light chain,             as shown in SEQ ID NO:69, or         -   h. a variable sequence of the heavy chain, as shown in SEQ             ID NO:80, and also a variable sequence of the light chain,             as shown in SEQ ID NO:79, or         -   i. a variable sequence of the heavy chain, as shown in SEQ             ID NO:90, and also a variable sequence of the light chain,             as shown in SEQ ID NO:89, or         -   j. a variable sequence of the heavy chain, as shown in SEQ             ID NO:100, and also a variable sequence of the light chain,             as shown in SEQ ID NO:99, or         -   k. a variable sequence of the heavy chain, as shown in SEQ             ID NO:110, and also a variable sequence of the light chain,             as shown in SEQ ID NO:109, or         -   l. a variable sequence of the heavy chain, as shown in SEQ             ID NO:120, and also a variable sequence of the light chain,             as shown in SEQ ID NO:119.     -   6. The antibody according to any of the preceding embodiments         which is an IgG antibody.     -   7. The antibody according to any of the preceding embodiments,         comprising:         -   a. a sequence of the heavy chain, as shown in SEQ ID NO:2,             and also a sequence of the light chain, as shown in SEQ ID             NO:1, or         -   b. a sequence of the heavy chain, as shown in SEQ ID NO:12,             and also a sequence of the light chain, as shown in SEQ ID             NO:11, or         -   c. a sequence of the heavy chain, as shown in SEQ ID NO:22,             and also a sequence of the light chain, as shown in SEQ ID             NO:21, or         -   d. a sequence of the heavy chain, as shown in SEQ ID NO:32,             and also a sequence of the light chain, as shown in SEQ ID             NO:31, or         -   e. a sequence of the heavy chain, as shown in SEQ ID NO:42,             and also a sequence of the light chain, as shown in SEQ ID             NO:41, or         -   f. a sequence of the heavy chain, as shown in SEQ ID NO:52,             and also a sequence of the light chain, as shown in SEQ ID             NO:51, or         -   g. a sequence of the heavy chain, as shown in SEQ ID NO:62,             and also a sequence of the light chain, as shown in SEQ ID             NO:61, or         -   h. a sequence of the heavy chain, as shown in SEQ ID NO:72,             and also a sequence of the light chain, as shown in SEQ ID             NO:71, or         -   i. a sequence of the heavy chain, as shown in SEQ ID NO:82,             and also a sequence of the light chain, as shown in SEQ ID             NO:81, or         -   j. a sequence of the heavy chain, as shown in SEQ ID NO:92,             and also a sequence of the light chain, as shown in SEQ ID             NO:91, or         -   k. a sequence of the heavy chain, as shown in SEQ ID NO:102,             and also a sequence of the light chain, as shown in SEQ ID             NO:101, or         -   l. a sequence of the heavy chain, as shown in SEQ ID NO:112,             and also a sequence of the light chain, as shown in SEQ ID             NO:111.     -   8. The antigen-binding fragment according to any of the         preceding embodiments or an antigen-binding fragment of an         antibody according to any of the preceding embodiments which is         an scFv, Fab, Fab′ fragment or a F(ab′)2 fragment.     -   9. The antibody or the antigen-binding fragment according to any         of the preceding embodiments which is a monoclonal antibody or         an antigen-binding fragment thereof     -   10. The antibody or the antigen-binding fragment according to         any of the preceding embodiments which is a human, humanized or         chimeric antibody or an antigen-binding fragment.

Particular preference is given to the anti-TWEAKR antibody TPP-2090.

It is an embodiment of this invention to provide antibodies suitable for transglutaminase (TGase)-mediated conjugation of a kinesin spindle protein inhibitor.

Wild-type full-length IgG antibodies of human isotype possess a conserved acceptor glutamine at residue 295 (Kabat EU numbering) of the heavy chain which is accessible and reactive in presence of a TGase, to form a conjugate from the antibody and the suitable compound, when the antibody is in a non-glycosylated form. Such an “aglycosyl antibody” or “aglycosylated antibody” or “deglycosylated antibody” comprises an Fc region lacking the glycans attached to the conserved N-linked site in the CH₂ domains of the Fc region.

Aglycosyl antibodies can for example be generated by expressing the antibodies in expression systems lacking glycosylation. Aglycosyl antibodies may be prepared by expression the antibodies in a prokaryotic host. Suitable prokaryotic hosts for include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In another embodiment of this invention, aglycosyl antibodies may be achieved using mammalian expression systems together with the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983), Proc Natl Acad Sci USA, 80(21):6632-6). That is, the modification is the prevention of glycosylation at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody. In another embodiment of the invention the glycans attached to the conserved N-linked site in the CH2 domains of the Fc region antibody are removed, which means the antibody is deglycosylated. Methods for enzymatic deglycosylation of antibodies are well known in the art (e.g. Winkelhake & Nicolson (1976), J Biol Chem. 251(4):1074-80). Deglycosylated antibodies may e.g. be prepared by enzymatic deglycosylation using e.g. PNGase F.

In another embodiment of the invention aglycosylated antibodies are prepared by mutation of the heavy chain glycosylation site of N297 (using Kabat EU numbering). Enzymatic conjugation of such engineered aglycosylated antibodies was described for aglycosylated antibody variants bearing the mutations N297D, N297Q (Jeger et al., Angewandte Chemie Int. Ed. Engl 49, 9995-9997 (2010)), or N297S (see patent applications WO2013092998A1 and WO2013092983A2). Furthermore this invention shows that transglutaminase could efficiently catalyze conjugation to aglycosylated antibody variants bearing the mutation N297A (Kabat EU numbering).

Additional or alternative sites reactive in the presence of a TGase can be created by engineering the antibodies. The compounds of the invention include glutamine engineered antibodies where one or more amino acids of a wild-type or parent antibody are replaced with (substituted by) a glutamine amino acid, or where a glutamine residue, optionally together with other amino acid residues (e.g. a tag comprising the acceptor glutamine residue), is introduced or added to a wild-type or parent.

A single site mutation that provides a glutamine that is accessible to a TGase may yield more than one engineered glutamine residue that can be conjugated if the antibody comprises more than one engineered chain. For example, a single site mutation will yield two engineered glutamine residues in an IgG due to the dimeric nature of the IgG antibody.

The glutamine amino acid residues of an antibody that are reactive, in the presence of a TGase under suitable conditions may be located in the heavy chain, typically in the constant domain. In one embodiment, an asparagine at amino acid position 297 (Kabat EU numbering) is replace with a residue different from glutamine. Preferred are N297D, N297Q, N297S or N297A, highly preferred is N297A. The antibody will have a constant region with a N297X substitution. An antibody having a N297X substitution and a glutamine at residue 295 (Kabat EU numbering) will therefore have one acceptor glutamine and thus one conjugation sites per heavy chain. The complete IgG form will therefore have two conjugates per antibody.

The glutamine amino acid residues of an antibody that are reactive, in the presence of a TGase under suitable conditions may be located in the heavy chain, typically in the constant domain. In one embodiment, an asparagine at amino acid position 297 (Kabat EU numbering) is substituted with a glutamine residue. The antibody will have a constant region with a N297Q substitution. An antibody having a N297Q substitution and a glutamine at residue 295 (Kabat EU numbering) will therefore have two acceptor glutamines and thus two conjugation sites per heavy chain. The complete IgG form will therefore have four conjugates per antibody.

The glutamine amino acid residues of an antibody that are reactive, in the presence of a TGase under suitable conditions may be located in the heavy chain, typically in the constant domain. In one embodiment, an asparagine at amino acid position 297 (Kabat EU numbering) is substituted with a glutamine residue and at position 295 (Kabat EU numbering) the glutamine is replaced. The antibody will have a constant region with a N297Q and Q295X substitution. Preferred is a Q295N substitution. An antibody having a N297Q substitution and no glutamine at residue 295 (EU Numbering) will therefore have one acceptor glutamine and thus one conjugation sites per heavy chain. The complete IgG form will therefore have two conjugates per antibody.

Preferred antibodies suitable for transglutaminase (TGase)-mediated conjugation comprise a:

-   -   i. N297X substitution wherein X is any amino acid but         asparagine; even more preferred are N297D, N297Q, N297S or         N297A, highly preferred are N297A and N297Q.     -   ii. N297Q substitution and a Q295X substitution wherein X is any         amino acid but glutamine, preferred is Q295N.

An advantageous approach for preparing conjugated antibodies will thus involve providing as starting materials antibodies lacking N297-linked glycosylation (such N-linked glycosylation interferes with TGase coupling).

Isotopes, Salts, Solvates, Isotopic Variants

The present invention also encompasses all suitable isotopic variants of the compounds according to the invention. An isotopic variant of a compound according to the invention is understood here as meaning a compound in which at least one atom within the compound according to the invention has been exchanged for another atom of the same atomic number, but with a different atomic mass than the atomic mass which usually or predominantly occurs in nature. Examples of isotopes which can be incorporated into a compound according to the invention are those of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine, such as ²H (deuterium), ³H (tritium) ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³²P, ³³P, ³³S, ³⁴S, ³⁵S, ³⁶S, ¹⁸F, ³⁶Cl, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁹I and ¹³¹I. Particular isotopic variants of a compound according to the invention, especially those in which one or more radioactive isotopes have been incorporated, may be beneficial, for example, for the examination of the mechanism of action or of the active compound distribution in the body; due to comparatively easy preparability and detectability, especially compounds labelled with ³H or ¹⁴C isotopes are suitable for this purpose. In addition, the incorporation of isotopes, for example of deuterium, can lead to particular therapeutic benefits as a consequence of greater metabolic stability of the compound, for example an extension of the half-life in the body or a reduction in the active dose required; such modifications of the compounds according to the invention may therefore in some cases also constitute a preferred embodiment of the present invention. Isotopic variants of the compounds according to the invention can be prepared by the processes known to those skilled in the art, for example by the methods described below and the procedures described in the working examples, by using corresponding isotopic modifications of the respective reagents and/or starting compounds.

Preferred salts in the context of the present invention are physiologically acceptable salts of the compounds according to the invention. Also encompassed are salts which are not themselves suitable for pharmaceutical applications but can be used, for example, for isolation or purification of the compounds according to the invention.

Physiologically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulphonic acids, for example salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid, naphthalenedisulphonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and benzoic acid.

Physiologically acceptable salts of the inventive compounds also include salts of conventional bases, by way of example and with preference alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts) and ammonium salts derived from ammonia or organic amines having 1 to 16 carbon atoms, by way of example and with preference ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysine and 1,2-ethylenediamine.

Solvates in the context of the invention are described as those forms of the compounds according to the invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of the solvates in which the coordination is with water. Solvates preferred in the context of the present invention are hydrates.

In addition, the present invention also encompasses prodrugs of the compounds according to the invention. The term “prodrugs” here denotes compounds which may themselves be biologically active or inactive, but are converted (for example by metabolic or hydrolytic means) to inventive compounds during their residence time in the body.

PARTICULAR EMBODIMENTS

The following embodiments are particularly preferred:

Embodiment A

An ADC (or APDC) of the formula BINDER

L-KSP]_(n) where KSP-L- is a compound of the formula (II), (IIIa), (IIIb), (IIIc), (IIId), (IIIe), any of (IV) to (IX), or the formula (IIf) below, the binder is an anti-TWEAKR antibody comprising an acceptor glutamine residue (particularly preferably an anti-TWEAKR antibody or a variant thereof which binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), especially the anti-TWEAKR antibody TPP-2090), aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid, and variants of the described antibodies being engineered to contain solvent accessible glutamine residues being substrates for bacterial transglutaminase, and n is 2 or 4

-   -   where         X₁ represents N, X₂ represents N and X₃ represents C;         X₁ represents CH, X₂ represents C and X₃ represents N;         X₁ represents NH, X₂ represents C and X₃ represents C; or         X₁ represents CH, X₂ represents N and X₃ represents C;         A represents C(═O);         R¹ represents -L-#1, —H, —COOH, —C(═O)—NHNH₂, —(CH₂)₁₋₃NH₂,         —C(═O)—NZ″(CH₂)₁₋₃NH₂ and —C(═O)—NZ″CH₂COOH, where Z″ represents         —H or —NH₂;         R² and R⁴ represent —H or -L-#1, or R² represents —H and R⁴         represents         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— (where P2         and Pe have the same meaning as defined above, e.g. as shown         with respect to formula (IIa)), or R² and R⁴ together (with         formation of a pyrrolidine ring) represent —CH₂—CHR¹⁰— or         —CHR¹⁰—CH₂—, where R¹⁰ represents —H or -L-#1;         R³ represents -L-#1 or a C₁₋₁₀-alkyl-, which may optionally be         substituted by —OH, —O-alkyl, —SH, —S-alkyl, —O—C(═O)—NH-alkyl,         —NH—C(═O)-alkyl, —NH—C(═O)—NH-alkyl, —S(═O)₂—NH-alkyl,         —NH-alkyl, —N(alkyl)₂ or —NH₂ (where alkyl is preferably         C₁₋₃-alkyl);         R⁵ represents -L-#1, H or F;         R⁶ and R⁷ independently of one another represent —H,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy or halogen (in particular —F, —Cl, —Br),         R⁸ represents a branched C₁₋₅-alkyl group which may be         substituted by -L-#1; and         R⁹ represents H or F,         where one of the substituents R¹, R², R³, R⁴, R⁵, R⁸ and R¹⁰         represents -L-#1, and         -L- represents the linker and #1 represents the bond to the         antibody,         and salts, solvates and salts of the solvates of the ADC.

The linker is preferably a linker § —(C═O)m-L1-L2-§ 517

-   -   where     -   m is 0 or 1;     -   § represents the bond to KSP and     -   § § represents the bond to the antibody, and     -   L2 represents:         #¹—(NH)_(p)—(C═O)_(q)-G4-NH—#² or         #¹—(NH)_(p)—(C═O)_(q)-G4-O—NH—#²     -   where     -   p is 0 or 1;     -   q is 0 or 1; and     -   G4 represents an optionally substituted alkyl or heteroalkyl         chain optionally wherein any carbon of the chain is substituted         with an alkoxy, hydroxyl, alkylcarbonyloxy, —S-alkyl, thiol,         —C(═O)—S-alkyl, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, amine,         —C(═O)—NH₂     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder,     -   and L1 is represented by formula         #1-(NR¹⁰)_(n)-(G1)_(o)-G3-^(#2)         where         R¹⁰ represents —H, —NH₂ or C₁-C₃-alkyl;         G1 represents NH—C(═O)— or

n is 0 or 1; o is 0 or 1; and G3 represents a bond or an optionally substituted linear or branched hydrocarbon chain having 1 to 100 carbon atoms from arylene groups and/or linear and/or branched and/or cyclic alkylene groups and which may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂, —NH—, —C(═O)—, —NH—C(═O)—, —C(═O)—NH—, —NMe-, —NHNH—, —S(═O)₂—NHNH—, —C(═O)—NHNH— and a 3- to 10-membered aromatic or non-aromatic heterocycle having UP to 4 heteroatoms selected from the group consisting of N, O and S, or —S(═O)— (preferably

where the side chains, if present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

Here, #¹ is the bond to the KSP inhibitor and #² is the bond to the coupling group to the binder (e.g. L2).

Embodiment B

An ADC of the formula BINDER

L-KSP]_(n) where KSP-L- is a compound of the formula (II), (IIa), (IIIb), (IIIc), (IIId), (IIIe), (IIf), any of (IV) to (IX) or of the formula (Hg) below, the binder is an antibody comprising an acceptor glutamine residue and n is 2 or 4:

-   -   where         X₁ represents N, X₂ represents N and X₃ represents C;         X₁ represents CH, X₂ represents C and X₃ represents N;         X₁ represents NH, X₂ represents C and X₃ represents C; or         X₁ represents CH, X₂ represents N and X₃ represents C;         A represents C(═O)—;         R¹ represents -L-#1, —H, —COOH, —C(═O)—NHNH₂, —(CH₂)₁₋₃NH₂,         —(C═O)—NZ″(CH₂)₁₋₃NH₂ and C(═O)—NZ″CH₂COOH, where Z″ represents         —H or —NH₂;         R² and R⁴ represent —H or -L-#1, or R² represents —H and R⁴         represents         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)— (where P2         and Pe have the same meaning as defined above, e.g. as shown         with respect to formula (IIa)), or R² and R⁴ together (with         formation of a pyrrolidine ring) represent —CH₂—CHR¹⁰— or         CHR¹⁰—CH₂—, where R¹⁰ represents —H or -L-#1;         R³ represents -L-#1 or a C₁₋₁₀-alkyl-, which may optionally be         substituted by —OH, —O-alkyl, —SH, —S-alkyl, —O—C(═O)-alkyl,         —O—C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —NH—C(═O)—NH-alkyl,         —S(═O)₂—NH-alkyl, —NH-alkyl, —N(alkyl)₂ or —NH₂ (where alkyl is         preferably C₁₋₃-alkyl);         R⁵ represents -L-#1, H or F;         R⁶ and R⁷ independently of one another represent —H,         C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy or halogen (in particular —F, —Cl, —Br),         R⁸ represents a branched C₁₋₅-alkyl group; and         R⁹ represents H or F,         where one of the substituents R¹, R², R³, R⁴, R⁵ and R¹⁰         represents -L-#1, and         -L- represents the linker and #1 represents the bond to the         antibody,         where -L- is represented by         § —(C═O)m-L1-L2-§ 517     -   where     -   m is 0 or 1;     -   § represents the bond to KSP and     -   § § represents the bond to the antibody, and     -   L2 represents:         #¹—(NH)_(p)—(C═O)_(q)-G4-NH—#² or         #¹—(NH)_(p)—(C═O)_(q)-G4-O—NH—#²     -   where     -   p is 0 or 1;     -   q is 0 or 1; and     -   G4 represents an optionally substituted alkyl or heteroalkyl         chain optionally wherein any carbon of the chain is substituted         with an alkoxy, hydroxyl, alkylcarbonyloxy, —S-alkyl, thiol,         —C(═O)—S-alkyl, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, amine,         —C(═O)—NH₂     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder,     -   and L1 is represented by formula         #¹—(NR¹⁹)_(n)-(G1)_(o)-G3-#²         where         R¹⁰ represents —H, —NH₂ or C₁-C₃-alkyl;         G1 represents NH—C(═O)— or

n is 0 or 1; o is 0 or 1; and G3 represents a bond or an optionally substituted linear or branched hydrocarbon chain having 1 to 100 carbon atoms from arylene groups and/or linear and/or branched and/or cyclic alkylene groups and which may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂, —NH—, —C(═O)—, —NH—C(═O)—, —C(═O)—NH—, —NMe-, —NHNH—, —S(═O)₂—NHNH—, —C(═O)—NHNH— and a 3- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from the group consisting of N, O and S, or —S(═O)— (preferably

where the side chains, if present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

#¹ is the bond to the KSP inhibitor and #² is the bond to the coupling group to the antibody (e.g. L2), and salts, solvates and salts of the solvates of the ADC,

Embodiment C

An ADC of the formula BINDER

L-KSP]_(n) where KSP-L- is a compound having the substructure I(sub) below, the binder is an anti-TWEAKR antibody comprising an acceptor glutamine residue (particularly preferably Trastuzumab or an anti-TWEAKR antibody which binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), especially the anti-TWEAK R antibody TPP-2090), anti-HER2 antibody or anti-EGRF antibody (preferably nimotuzumab), aglycosylated variants of these antibodies generated either by deglycosylation by PNGase F or by mutation of N297 (Kabat numbering) of the heavy chain to any amino acid, and variants of the described antibodies being engineered to contain one or more acceptor glutamine residues for transglutaminase (TGase) catalyzed reactions, and n is 2 or 4:

-   -   where     -   #a represents a bond to the remainder of the molecule;     -   R^(1a) represents -L-#1, —H or —(CH₂)₀₋₃Z, where Z represents         —H, halogen, —NHY³, —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R^(2a) and R^(4a) independently of one another represent -L-#1,         H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Z represents —H, halogen, —OY³, —SY³, NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NHC(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R^(2a) represents —H and     -   R^(4a) represents         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—         -   where P2 and P3 have the same meaning as defined above, e.g.             as shown with respect to formula (IIa),         -   where R²¹ represents H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group,             which may be substituted one or more times with —NH₂,             -   —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,                 N(alkyl)-C(═O)-alkyl, —SO₃H,             -   —S(═O)₂NH₂, —S(═O)₂—N(alkyl)₂, —COOH, —C(═O)—NH₂,                 —C(═O)—N(alkyl)₂, or —OH, or represent —H or a group                 —(O)_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1 and         -   where v is a number from 1 to 20,         -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂);     -   or     -   R^(2a) and R^(4a) together (with formation of a pyrrolidine         ring) represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, -L-#1, —NH₂, —SO₃H, —COOH, —SH,             —SO₃H or —OH,     -   where one of the substituents R^(1a), R^(2a), R^(4a) or R¹⁰         represents -L-#1,     -   -L- represents the linker and #1 represents the bond to the         antibody,     -   where -L- is represented by         § —(C═O)m-L1-L2-§ 517     -   where     -   m is 0 or 1;     -   § represents the bond to KSP and     -   § § represents the bond to the antibody, and     -   L2 represents #¹—(NH)_(p)—(C═O)_(q)-G4-NH—#² or         #¹—(NH)_(p)—(C═O)_(q)-G4-O—NH—#²     -   where     -   p is 0 or 1;     -   q is 0 or 1; and     -   G4 represents an optionally substituted alkyl or heteroalkyl         chain optionally wherein any carbon of the chain is substituted         with an alkoxy, hydroxyl, alkylcarbonyloxy, —S-alkyl, thiol,         —C(═O)—S-alkyl, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, amine,         —C(═O)—NH₂     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder,     -   and L1 is represented by formula         #1-(NR¹⁰)_(n)-(G1)_(o)-G3-^(#2)         where         R¹⁰ represents —H, —NH₂ or C₁-C₃-alkyl;         G1 represents NH—C(═O)— or

n is 0 or 1; o is 0 or 1; and G3 represents a bond or an optionally substituted linear or branched hydrocarbon chain having 1 to 100 carbon atoms from arylene groups and/or linear and/or branched and/or cyclic alkylene groups and which may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂, —NH—, —C(═O)—, —NH—C(═O)—, —C(═O)—NH—, —NMe-, —NHNH—, —S(═O)₂—NHNH—, —C(═O)—NHNH— and a 3- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from the group consisting of N, O and S, or —S(═O)— (preferably

where the side chains, if present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

Here, #¹ is the bond to the KSP inhibitor and #² is the bond to the coupling group to the binder (e.g. L2). and salts, solvates and salts of the solvates of the ADC.

Embodiment D

An ADC of the formula BINDER

L-KSP]_(n) where KSP-L- is a compound of the formula (II), (IIa), (IIIb), (IIIc), (IIId), (IIe), (IIg), any of (III) to (IX), or of the formula (IIh) below, the binder is an antibody comprising an acceptor glutamine residue and n is a number 2 or 4:

-   -   where

-   X₁ represents N, X₂ represents N and X₃ represents C;

-   X₁ represents CH, X₂ represents C and X₃ represents N;

-   X₁ represents NH, X₂ represents C and X₃ represents C; or

-   X₁ represents CH, X₂ represents N and X₃ represents C;

-   A represents —C(═O)—;

-   R¹ represents -L-#1;

-   R² and R⁴ represent —H,     -   or

-   R² represents —H and

-   R⁴ represents R²¹—C(═O)—P3₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—     -   where P2 and P3 have the same meaning as defined above, e.g. as         shown with respect to formula (IIa),     -   or

-   R² and R⁴ together (with formation of a pyrrolidine ring) represent     —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—, where R¹⁰ represents H;

-   R³ represents C₁₋₁₀-alkyl, which may be substituted by 1-3 —OH     groups, 1-3 —O-alkyl groups, 1-3 —SH groups, 1-3 —S-alkyl groups,     1-3 —O—C(═O)-alkyl groups, 1-3 —O—C(═O)—NH-alkyl groups, 1-3     —NH—C(═O)-alkyl groups, 1-3 —NH—C(═O)—NH-alkyl groups, 1-3     —S(═O)_(n)-alkyl groups, 1-3 —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl     groups, 1-3 —N(alkyl)₂ groups or 1-3 —NH₂ groups,     -   where alkyl is preferably C₁₋₃-alkyl or -MOD     -   where -MOD represents —(NR¹⁰)_(n)-(G1)_(o)-G2-H,     -   where     -   R¹⁰ represents —H or C₁-C₃-alkyl;         -   where G1 represents NH—C(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents NH—C(═O)— or

-   -   -    R¹⁰ does not represent NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1; and         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,             —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—,             —C(═O)—NR^(y)NR^(y)—,         -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid, or             represents —C(═O)—, —CR^(x)═N—O—         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,         -   where the hydrocarbon chain including any side chains may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   where -MOD preferably has at least one group —COOH;

-   R⁵ represents H or F;

-   R⁶ and R⁷ independently of one another represent —H, C₁₋₃-alkyl,     fluoro-C₁₋₃-alkyl, C₂₋₄-alkenyl, fluoro-C₂₋₄-alkenyl, C₂₋₄-alkynyl,     fluoro-C₂₋₄-alkynyl, hydroxy or halogen (in particular —F, —Cl,     —Br),

-   R⁸ represents a branched C₁₋₅-alkyl group; and

-   R⁹ represents H or F,

-   where -L- represents the linker and #1 represents the bond to the     antibody,

-   where -L- is represented by     § —(C═O)m-L1-L2-§ 517     -   where     -   m is 0 or 1;     -   § represents the bond to KSP and     -   § § represents the bond to the antibody, and     -   L2 represents:         #¹—(NH)_(p)—(C═O)_(q)-G4-NH—#² or         #¹—(NH)_(p)—(C═O)_(q)-G4-O—NH—#²     -   where     -   p is 0 or 1;     -   q is 0 or 1; and     -   G4 represents an optionally substituted alkyl or heteroalkyl         chain optionally wherein any carbon of the chain is substituted         with an alkoxy, hydroxyl, alkylcarbonyloxy, —S-alkyl, thiol,         —C(═O)—S-alkyl, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, amine,         —C(═O)—NH₂     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder,     -   and L1 is represented by formula         #1-(NR¹⁰)_(n)-(G1)_(o)-G3-^(#2)

-   where

-   R¹⁰ represents —H, —NH₂ or C₁-C₃-alkyl;

-   G1 represents NH—C(═O)— or

-   n is 0 or 1; -   o is 0 or 1; and -   G3 represents a bond or an optionally substituted linear or branched     hydrocarbon chain having 1 to 100 carbon atoms from arylene groups     and/or linear and/or branched and/or cyclic alkylene groups and     which may be interrupted once or more than once by one or more of     the groups —O—, —S—, —S(═O)—, —S(═O)₂, —NH—, —C(═O)—, —NH—C(═O)—,     —C(═O)—NH—, —NMe-, —NHNH—, —S(═O)₂—NHNH—, —C(═O)—NHNH— and a 3- to     10-membered aromatic or non-aromatic heterocycle having up to 4     heteroatoms selected from the group consisting of N, O and S, or     —S(═O)— (preferably

where where the hydrocarbon chain including the side chains, if present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, —NH—CNNH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid.

-   where #¹ is the bond to the KSP inhibitor and #² is the bond to the     coupling group to the antibody (e.g. L2), -   and salts, solvates, salts of the solvates and epimers of the ADC.

Embodiment E

A site specific and homogenous ADC of the formula BINDER

L-KSP]_(n) where KSP-L- is a compound of the formula (II), (IIa), (IIIb), (IIIc), (IIId), (IIe), (IIf), (IIg), or any of (III) to (IX), or of the formula (IIh) and salts, solvates and salts of the solvates of the ADC.

One embodiment of the invention is a conjugate of a binder or derivative thereof with one or more active compound molecules, the active compound molecule being a kinesin spindle protein inhibitor attached to the binder via a linker L, where the linker L is attached to a glutamine side chain of the binder, where 1 to 5 kinesin spindle protein inhibitors are attached to the linker L, where the kinesin spindle protein inhibitor has the formula (IIa) below:

-   -   where     -   X₁ represents N, X₂ represents N and X₃ represents C; or     -   X₁ represents N, X₂ represents C and X₃ represents N; or     -   X₁ represents CH or CF, X₂ represents C and X₃ represents N; or     -   X₁ represents NH, X₂ represents C and X₃ represents C; or     -   X₁ represents CH, X₂ represents N and X₃ represents C     -   (with X₁ representing CH, X₂ representing C and X₃ representing         N being preferred);     -   R¹ represents —H, -MOD, L-#1 or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂;     -   R² represents -L-#1, H, -MOD, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where -MOD is represented as defined infra,         -   where Z represents —H, halogen, —OY³, —SY³, NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′, and         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NHC(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R⁴ represents -L-#1, —H, —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂, and         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C1-6-alkyl,     -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents -L-#1, —H, —NH₂, —SO₃H, —COOH, —SH or             —OH,     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         C(═N—NH₂)—;     -   R³ represents -L-#1, -MOD or an optionally substituted alkyl,         cycloalkyl, aryl, heteroaryl, heteroalkyl, heterocycloalkyl         group, preferably -L-#1 or a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or         C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)_(n)-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where n represents 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′ and         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NHC(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′, where         -   where Z′ represents —H, —SO₃H, —NH₂ or COOH;     -   R⁵ represents —H, —NH₂, —NO₂, halogen (in particular —F, —Cl,         —Br), —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH     -   R⁶ and R⁷ independently of one another represent —H, cyano,         GAD-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         hydroxy, —NO₂, —NH₂, —COOH or halogen (in particular —F, —Cl,         —Br),     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl,         fluoro-C₂₋₁₀-alkenyl, C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl,         C₄₋₁₀-cycloalkyl, fluoro-C₄₋₁₀-cycloalkyl or —(CH₂)₀₋₂—(HZ²),         -   where HZ² represents a 4- to 7-membered heterocycle having             up to two heteroatoms selected from the group consisting of             N, O and S (preferably oxetane), where each of these groups             may be substituted by —OH, —COOH, —NH₂ or -L-#1;     -   where one or none of the substituents R¹, R², R³, R⁴ R⁵, R⁸ and         R¹⁰ represents or (in the case of R⁸) contains L-#1,     -   where L represents the linker and     -   where #1 represents the bond to the binder or derivative         thereof,     -   where -MOD represents —(NR¹⁰)_(n)-(G1)_(o)-G2-H,         -   where R¹⁰ represents —H or C₁-C₃-alkyl;         -   where G1 represents —NH—C(═O)—, —C(═O)—NH— or

-   -   -    (where, if G1 represents NH—C(═O)— or

-   -   -    R¹⁰ does not represent NH₂);         -   where n is 0 or 1;         -   where o is 0 or 1; and         -   where G2 represents a linear and/or branched hydrocarbon             group which has 1 to 10 carbon atoms and which may be             interrupted once or more than once by one or more of the             groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—,             —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—,             —C(═O)—NR^(y)NR^(y)—,         -   where R^(y) represents —H, phenyl, C₁-C₁₀-alkyl,             C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid, or             represents —C(═O)—, —CR^(x)═N—O—         -   where R^(x) represents —H, C₁-C₃-alkyl or phenyl,         -   where the hydrocarbon chain including any side chains may be             substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂,             sulphonamide, sulphone, sulphoxide or sulphonic acid,         -   where G3 represents —H or —COOH, and         -   where -MOD preferably has at least one group —COOH;

    -   and the salts, solvates, salts of the solvates, and epimers         thereof.

Another embodiment of the invention is a conjugate as defined above where X₁ represents CH, X₂ represents C and X₃ represents N.

Another embodiment of the invention is a conjugate as defined above where the substituent R¹ represents -L-#1.

Another embodiment of the invention is a conjugate of a binder or derivative thereof with the active compound molecule being a kinesin spindle protein inhibitor attached to the binder via a linker L, where the linker L is attached to a glutamine side chain of the binder, where the kinesin spindle protein inhibitor has the substructure below:

-   -   where     -   #a represents a bond to the rest of the molecule;     -   R^(1a) represents H or —(CH₂)₀₋₃Z,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ (e.g. —(CH₂)₀₋₃Z′) or             —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R^(2a) and R^(4a) independently of one another represent —H,         —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z, or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, —NH₂, —SO₃H, —COOH, —SH, or —OH;         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   where the kinesin spindle protein inhibitor is attached to the         linker by substitution of a hydrogen atom at R^(1a), R^(2a),         R^(4a) or at the pyrrolidine ring formed by R^(2a) and R^(4a),     -   and the salts, solvates, salts of the solvates, and epimers         thereof.

Another embodiment of the invention is a conjugate as defined above where the kinesin spindle protein inhibitor is represented by general formula (I):

-   -   where     -   R^(1a) represents —H or —(CH₂)₀₋₃Z,         -   where Z represents —H, —NHY³, —OY³, —SY³, halogen,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ or —CH(CH₂W)Z′, and         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—(C═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂;     -   R^(2a) and R^(4a) independently of one another represent —H,         —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   or     -   R^(2a) and R^(4a) together (with formation of a pyrrolidine         ring) represent CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents —H, —NH₂, —SO₃H, —COOH, —SH, or —OH;         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by —NH—C(═O)—NH₂, or represents aryl             or benzyl which are optionally substituted by —NH₂,         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl;     -   R^(3a) represents an optionally substituted alkyl, cycloalkyl,         aryl, heteroaryl, heteroalkyl, heterocycloalkyl group,         preferably -L-BINDER, or a C₁₋₁₀-alkyl, C₆₋₁₀-aryl or         C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl, C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or         C₅₋₁₀-heterocycloalkyl group which may be substituted by 1-3 —OH         groups, 1-3 halogen atoms, 1-3 halogenated alkyl groups (each         having 1-3 halogen atoms), 1-3 —O-alkyl groups, 1-3 —SH groups,         1-3 —S-alkyl groups, 1-3 —O—C(═O)-alkyl groups, 1-3         —O—C(═O)—NH-alkyl groups, 1-3 —NH—C(═O)-alkyl groups, 1-3         —NH—C(═O)—NH-alkyl groups, 1-3 —S(═O)n-alkyl groups, 1-3         —S(═O)₂—NH-alkyl groups, 1-3 —NH-alkyl groups, 1-3 —N(alkyl)₂         groups, 1-3 —NH₂ groups or 1-3 —(CH₂)₀₋₃Z groups,         -   where n is 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NH—C(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH     -   where “alkyl” preferably represents C₁₋₁₀-alkyl);     -   R^(8a) represents C₁₋₁₀-alkyl;     -   HZ represents a mono- or bicyclic heterocycle which may be         substituted by one or more substituents selected from the group         consisting of halogen, C₁₋₁₀-alkyl groups, C₆₋₁₀-aryl groups and         C₆₋₁₀-aralkyl groups which may optionally be substituted by         halogen;     -   where the kinesin spindle protein inhibitor is attached to the         linker by substitution of a hydrogen atom at R^(1a), R^(2a),         R^(3a), R^(4a), R^(8a) or at the pyrrolidine ring formed by         R^(2a) and R^(4a),     -   and the salts, solvates and salts of the solvates thereof.

Another embodiment of the invention is a conjugate as defined above, where the linker L is attached to a glutamine side chain of the binder, where 1 to 5 kinesin spindle protein inhibitors are attached to the linker L, where the active compound molecule linker is represented by general formula (II):

-   -   where     -   X₁ represents N, X₂ represents N and X₃ represents C; or     -   X₁ represents N, X₂ represents C and X₃ represents N; or     -   X₁ represents CH, X₂ represents C and X₃ represents N; or     -   X₁ represents NH, X₂ represents C and X₃ represents C; or     -   X₁ represents CH, X₂ represents N and X₃ represents C     -   R¹ represents —H, -L-#1 or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′             -   (e.g. —(CH₂)₀₋₃Z′) or —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —SO₃H, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂;     -   R² and R⁴ independently of one another represents -L-#1, —H,         —C(═O)—CHY⁴—NHY⁵ or —(CH₂)₀₋₃Z,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂, and         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   or     -   R² represents —H and     -   R⁴ represents represents a group of         R²¹—(C═O)₍₀₋₁₎—(P3)₍₀₋₂₎-P2-NH—CH(CH₂C(═O)—NH₂)—C(═O)—         -   where R²¹ represents H, C₁₋₁₀-alkyl-, C₅₋₁₀-aryl- or             C₆₋₁₀-aralkyl-, C₅₋₁₀-heteroalkyl-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl-, C₅₋₁₀-heterocycloalkyl-,             heteroaryl-, heteroaryl-alkyl-, C₁₋₁₀-alkoxy-,             C₆₋₁₀-aryloxy- or C₆₋₁₀-aralkoxy-, C₅₋₁₀-heteroalkoxy-,             C₁₋₁₀-alkyl-O—C₆₋₁₀-aryloxy-, C₅₋₁₀-heterocycloalkoxy group,             which may be substituted one or more times with —NH₂,             -   —NH-alkyl, —N(alkyl)₂, —NH—C(═O)-alkyl,                 N(alkyl)-C(═O)-alkyl, —SO₃H,             -   —S(═O)₂NH₂, —S(═O)₂—N(alkyl)₂, —COOH, —C(═O)—NH₂,                 —C(═O)—N(alkyl)₂, or —OH, or represent —H or a group                 —(O)_(x)—(CH₂CH₂O)_(y)—R²²,         -   where x is 0 or 1 and         -   where v is a number from 1 to 20,         -   where R²² represents —H, -alkyl (preferably C₁₋₁₂-alkyl),             —CH₂—COOH, —CH₂—CH₂—COOH, or —CH₂—CH₂—NH₂);         -   where P2 and P3 have the same meaning as defined above, e.g.             as shown with respect to formula (IIa),     -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent —CH₂—CHR¹⁰— or —CHR¹⁰—CH₂—,         -   where R¹⁰ represents L-#1, —H, —NH₂, —SO₃H, —COOH, —SH or             —OH,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   A represents —C(═O)—, —S(═O)—, —S(═O)₂—, —S(═O)₂—NH— or         C(═N—NH₂)—;     -   R³ represents -L-#1 or an optionally substituted alkyl, aryl,         heteroaryl, or heterocycloalkyl group, preferably -L-#1 or a         C₁₋₁₀-alkyl, C₆₋₁₀-aryl, C₆₋₁₀-aralkyl, C₅₋₁₀-heteroalkyl,         C₁₋₁₀-alkyl-O—C₆₋₁₀-aryl or C₅₋₁₀-heterocycloalkyl group which         may be substituted by 1-3 —OH groups, 1-3 halogen atoms, 1-3         halogenated alkyl groups (each having 1-3 halogen atoms), 1-3         —O-alkyl groups, 1-3 —SH groups, 1-3 —S-alkyl groups, 1-3         —O—C(═O)-alkyl groups, 1-3 —O—C(═O)—NH-alkyl groups, 1-3         —NH—C(═O)-alkyl groups, 1-3 —NH—C(═O)—NH-alkyl groups, 1-3         —S(═O)_(n)-alkyl groups, 1-3 —S(═O)₂—NH-alkyl groups, 1-3         —NH-alkyl groups, 1-3 —N(alkyl)₂ groups, 1-3 —NH₂ groups or 1-3         —(CH₂)₀₋₃Z groups,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof,         -   where n represents 0, 1 or 2,         -   where Z represents —H, halogen, —OY³, —SY³, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′ and         -   where Y³ represents —H, —(CH₂)₀₋₃—CH(NHC(═O)—CH₃)Z′,             —(CH₂)₀₋₃—CH(NH₂)Z′ or —(CH₂)₀₋₃Z′, where         -   where Z′ represents —H, —SO₃H, —NH₂ or COOH;     -   R⁵ represents -L-#1, —H, —F, —NH₂, —NO₂, halogen (in particular         —F, —Cl, —Br), —SH or —(CH₂)₀₋₃Z,         -   where Z represents —H, —OY³, —SY³, halogen, —NHY³,             —C(═O)—NY¹Y² or —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂ or —(CH₂)₀₋₃Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —SO₃H, —NH₂ or —COOH;     -   where one of the substituents R¹, R², R³, R⁴ and R⁵ represents         -L-#1,     -   L represents the linker and #1 represents the bond to the binder         or derivative thereof,     -   R⁶ and R⁷ independently of one another represent —H, cyano,         fluoro-C₁₋₁₀-alkyl, C₂₋₁₀-alkenyl, fluoro-C₂₋₁₀-alkenyl,         C₂₋₁₀-alkynyl, fluoro-C₂₋₁₀-alkynyl, hydroxy or halogen (in         particular —F, —Cl, —Br),     -   R⁸ represents C₁₋₁₀-alkyl, fluoro-C₁₋₁₀-alkyl, C₄₋₁₀-cycloalkyl,         fluoro-C₄₋₁₀-cycloalkyl, or optionally substituted oxetane; and     -   R⁹ represents —H, —F, —CH₃, —CF₃, —CH₂F or —CHF₂;     -   and the salts, solvates, salts of the solvates, and epimers         thereof.

Another embodiment of the invention is a conjugate as defined above,

-   -   R¹ represents —H, -L-#1 or —(CH₂)₀₋₃Z,         -   where -L-#1 represents the linker and #1 represents the bond             to the binder or derivative thereof         -   where Z represents —H, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or             —C(═O)—OY³,         -   where Y¹ and Y² independently of one another represent —H,             —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ or —CH(CH₂W)Z′,         -   where Y³ represents —H or —(CH₂)₀₋₃Z′,         -   where Z′ represents —H, —NH₂, —COOH,             —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH,         -   where W represents —H or —OH,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂;     -   R² and R⁴ independently of one another represent -L-#1, —H or         —C(═O)—CHY⁴—NHY⁵,     -   or     -   R² and R⁴ together (with formation of a pyrrolidine ring)         represent —CH₂—CHR¹⁰—,         -   where R¹⁰ represents —H, -L-#1, —NH₂, —COOH, —SH, —OH or             —SO₃H,         -   where Y⁴ represents linear or branched C₁₋₆-alkyl which is             optionally substituted by             -   —NH—C(═O)—NH₂, or represents aryl or benzyl which are                 optionally substituted by —NH₂, and         -   where Y⁵ represents —H or —C(═O)—CHY⁶—NH₂,         -   where Y⁶ represents linear or branched C₁₋₆-alkyl,     -   A represents —C(═O)—,     -   R³ represents —(CH₂)OH or -L-#1, and     -   R⁵ represents -L-#1 or —H,     -   where one of the substituents R¹, R², R³, R⁴ and R⁵ represents         -L-#1,     -   and the salts, solvates, salts of the solvates, and epimers         thereof.

Another embodiment of the invention is a conjugate as defined above where R⁶ and R⁷ independently of one another represent —H, C₁₋₃-alkyl or halogen.

Another embodiment of the invention is a conjugate as defined above where R⁸ represents C₁₋₄-alkyl (preferably tert-butyl).

Another embodiment of the invention is a conjugate as defined above where R⁹ represents —H.

Another embodiment of the invention is a conjugate as defined above where R⁶ and R⁷ represent —F.

Another embodiment of the invention is a conjugate as defined above where the binder or derivative thereof is a binder peptide or protein or a derivative of a binder peptide or protein.

Another embodiment of the invention is a conjugate as defined above where the conjugate has 2 conjugation sites per binder.

Another embodiment of the invention is a conjugate according as defined above where the conjugate has 4 conjugation sites per binder.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein represents an antibody or the derivative of the binder peptide or protein comprising an acceptor glutamine side chain which can be recognized by transglutaminase.

Another embodiment of the invention is a conjugate as defined above produced by transglutaminase mediated conjugation.

Another embodiment of the invention is a conjugate as defined above produced using Transglutaminase originated from Streptomyces Mobaraensis.

Another embodiment of the invention is a conjugate as defined above where the binder binds to a cancer target molecule.

Another embodiment of the invention is a conjugate as defined above where the binder binds to an extracellular target molecule.

Another embodiment of the invention is a conjugate as defined above where the binder, after binding to the extracellular target molecule, is internalized and processed intracellularly (preferably lysosomally) by the cell expressing the target molecule.

Another embodiment of the invention is a conjugate s defined above where the binder peptide or protein is a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein is an antibody bearing acceptor glutamine residue(s) in its heavy chain, optionally within the CH2 domain.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein is an antibody bearing acceptor glutamine residue is in its heavy chain at position 295 (KABAT numbering system).

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein is an antibody comprising a N297X substitution wherein X is any amino acid but asparagine; even more preferred are N297D, N297Q, N297S or N297A, highly preferred are N297A and N297Q.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein is an antibody comprising N297Q substitution and a Q295X substitution wherein X is any amino acid but glutamine, preferred is Q295N.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein is an antibody comprising an asparagine at residue 297 that substantially lacks N-linked glycosylation.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein is an antibody produced in a host cell that produces antibodies lacking N-linked glycosylation at amino acid residue N297.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein is an anti-HER2 antibody, an anti-EGFR antibody, an anti-TWEAKR antibody or an antigen-binding fragment thereof.

Another embodiment of the invention is a conjugate as defined above where the anti-TWEAKR antibody binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), preferably the anti-TWEAKR antibody TPP2090 and aglycosylated variants thereof.

Another embodiment of the invention is a conjugate as defined above where the anti-TWEAKR antibody binds specifically to amino acid D in position 47 (D47) of TWEAKR (SEQ ID NO:169), preferably the anti-TWEAKR antibody TPP-2090-HC-N297A or TPP-2090-HC-N297Q.

Another embodiment of the invention is a conjugate as defined above, where the linker L is attached to a

glutamine side chain of the binder, where 1 to 5 kinesin spindle protein inhibitors are attached to the linker L, where the linker L has one of the basic structures (i) to (iv) below:

-   -   (i) —(C═O)_(m)-SG1-L1-L2-     -   (ii) —(C═O)_(m)-L1-SG-L1-L2-     -   (iii) —(C═O)_(m)-L1-L2-     -   (iv) —(C═O)_(m)-L1-SG-L2     -   where m is 0 or 1, SG and SG1 are in vivo cleavable groups, L1         independently of one another represent organic groups not         cleavable in vivo, and L2 represents a coupling group to the         binder.

Another embodiment of the invention is a conjugate as defined above, where the linker L is attached to a glutamine side chain of the binder, where 1 to 5 kinesin spindle protein inhibitors are attached to the linker L, where the in vivo cleavable group SG is a 2-8 oligopeptide group, preferably dipeptide group or a disulphide, a hydrazone, an acetal or an aminal and SG1 is a 2-8 oligopeptide group, preferably a dipeptide group.

Another embodiment of the invention is a conjugate as defined above where the linker is attached to a glutamine side chain and has the formula below: § —(C═O)m-L1-L2-§ 517

-   -   where     -   m is 0 or 1;     -   § represents the bond to the active compound molecule and     -   § § represents the bond to the binder peptide or protein, and     -   L1 represents —(NR¹⁰)n-(G1)o-G3-,     -   where R¹⁰ represents —H, —H₂ or C₁-C₃-alkyl;     -   G1 represents —NH—C(═O)— or

-   -   n is 0 or 1;     -   o is 0 or 1; and     -   G3 represents a bond or an optionally substituted linear or         branched hydrocarbon chain having 1 to 100 carbon atoms from         arylene groups and/or linear and/or branched and/or cyclic         alkylene groups and which may be interrupted once or more than         once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂,         —NH—, —C(═O)—, —NH—C(═O)—, —C(═O)—NH—, —NMe-, —NHNH—,         —S(═O)₂—NHNH—, —C(═O)—NHNH— and a 3- to 10-membered aromatic or         non-aromatic heterocycle having up to 4 heteroatoms selected         from the group consisting of N, O and S, or —S(═O)— (preferably

-   -    where where the hydrocarbon chain including the side chains, if         present, may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂,         —NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic         acid.     -   or represents one of the groups below:

-   -   where Rx represents —H, C₁-C₃-alkyl or phenyl.     -   L2 represents #¹—(NH)_(p)—(C═O)_(q)-G4-NH—#² or         #¹—(NH)_(p)—(C═O)_(q)-G4-O—NH—#²     -   where     -   p is 0 or 1;     -   q is 0 or 1; and     -   G4 represents an optionally substituted alkyl or heteroalkyl         chain optionally wherein any carbon of the chain is substituted         with an alkoxy, hydroxyl, alkylcarbonyloxy, —S-alkyl, thiol,         —C(═O)—S-alkyl, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, amine,         —C(═O)—NH₂     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder,

Another embodiment of the invention is a conjugate as defined above where the hydrocarbon chain is interrupted by one of the groups below:

-   -   where X represents —H or a C₁₋₁₀-alkyl group which may         optionally be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂,         —NH—CN—NH₂, sulphone, sulphoxide or sulphonic acid.

Another embodiment of the invention is a conjugate as defined above where L2 is one of the groups below:

-   -   L2 is

-   -   with Ry is —H, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —C(═O)—NH₂, or         —NH₂.     -   where     -   #¹ denotes the point of attachment to group L¹,     -   #² denotes the point of attachment to the glutamine residue of         the binder.

Another embodiment of the invention is a conjugate as defined above where Ry is H or NHCOMe

Another embodiment of the invention is a conjugate as defined above where R¹ or R⁴ represents -L-#1.

Another embodiment of the invention is a conjugate as defined above where the anti-TWEAKR antibody is an agonistic antibody.

Another embodiment of the invention is a conjugate as defined above which comprises:

-   -   a variable heavy chain comprising:     -   a. a CDR1 of the heavy chain encoded by an amino acid sequence         comprising the formula PYPMX (SEQ ID NO: 171), where X is I or         M;     -   b. a CDR2 of the heavy chain encoded by an amino acid sequence         comprising the formula YISPSGGXTHYADSVKG (SEQ ID NO: 172), where         X is S or K; and     -   c. a CDR3 of the heavy chain encoded by an amino acid sequence         comprising the formula GGDTYFDYFDY (SEQ ID NO: 173);         -   and a variable light chain comprising:     -   d. a CDR1 of the light chain encoded by an amino acid sequence         comprising the formula RASQSISXYLN (SEQ ID NO: 174), where X is         G or S;     -   e. a CDR2 of the light chain encoded by an amino acid sequence         comprising the formula XASSLQS (SEQ ID NO: 175), where X is Q, A         or N; and     -   f. a CDR3 of the light chain encoded by an amino acid sequence         comprising the formula QQSYXXPXIT (SEQ ID NO: 176), where X at         position 5 is T or S, X at position 6 is T or S and X at         position 8 is G or F.

Another embodiment of the invention is a conjugate as defined above which comprises:

-   -   a. a variable sequence of the heavy chain, as shown in SEQ ID         NO:10, and also a variable sequence of the light chain, as shown         in SEQ ID NO:9, or     -   b. a variable sequence of the heavy chain, as shown in SEQ ID         NO:20, and also a variable sequence of the light chain, as shown         in SEQ ID NO:19, or     -   c. a variable sequence of the heavy chain, as shown in SEQ ID         NO:30, and also a variable sequence of the light chain, as shown         in SEQ ID NO:29, or     -   d. a variable sequence of the heavy chain, as shown in SEQ ID         NO:40, and also a variable sequence of the light chain, as shown         in SEQ ID NO:39, or     -   e. a variable sequence of the heavy chain, as shown in SEQ ID         NO:50, and also a variable sequence of the light chain, as shown         in SEQ ID NO:49, or     -   f. a variable sequence of the heavy chain, as shown in SEQ ID         NO:60, and also a variable sequence of the light chain, as shown         in SEQ ID NO:59, or     -   g. a variable sequence of the heavy chain, as shown in SEQ ID         NO:70, and also a variable sequence of the light chain, as shown         in SEQ ID NO:69, or     -   h. a variable sequence of the heavy chain, as shown in SEQ ID         NO:80, and also a variable sequence of the light chain, as shown         in SEQ ID NO:79, or     -   i. a variable sequence of the heavy chain, as shown in SEQ ID         NO:90, and also a variable sequence of the light chain, as shown         in SEQ ID NO:89, or     -   j. a variable sequence of the heavy chain, as shown in SEQ ID         NO:100, and also a variable sequence of the light chain, as         shown in SEQ ID NO:99, or     -   k. a variable sequence of the heavy chain, as shown in SEQ ID         NO:110, and also a variable sequence of the light chain, as         shown in SEQ ID NO:109, or     -   l. a variable sequence of the heavy chain, as shown in SEQ ID         NO:120, and also a variable sequence of the light chain, as         shown in SEQ ID NO:119.

Another embodiment of the invention is a conjugate as defined above where the antibody is an IgG antibody.

Another embodiment of the invention is a process for preparing the conjugate as defined above where a compound of one of the formulae below, preferably in the form of its trifluoroacetic acid salt, is conjugated to a residue of a binder peptide or protein using transglutaminase, where the compound is preferably employed in a 2- to 100-fold molar excess with respect to the binder peptide or protein:

-   -   where X₁, X₂, X₃, SG, L1, R1 have the same meaning as above,         e.g. in Formula (IIa), and Ry is —H, —C(═O)—NH-alkyl,         —NH—C(═O)-alkyl, —C(═O)—NH₂, or —NH₂.

-   -   where X₁, X₂, X₃, SG, L1, R1 and R^(x) have the same meaning as         defined above.

Another embodiment of the invention is a conjugate as defined above where the binder peptide or protein represents an antibody or the derivative of the binder peptide or protein according to the following formula

Another embodiment of the invention is a pharmaceutical composition comprising a conjugate as defined above or a compound as defined above in combination with an inert non-toxic pharmaceutically suitable auxiliary.

Another embodiment of the invention is a conjugate as defined above or compound as defined above for use in a method for the treatment and/or prophylaxis of diseases.

Another embodiment of the invention is a conjugate as defined above or compound as defined above for use in a method for the treatment of hyperproliferative and/or angiogenic disorders.

Another embodiment of the invention is a method for the treatment and/or prophylaxis of hyperproliferative and/or angiogenic disorders in humans and animals using an effective amount of at least one conjugate as defined above or compound as defined above.

Particularly preferred embodiments are conjugates according to one of the following formulae, where Ak3a, Ak3b, AD3d, Ak3e, represent a binder, preferably an antibody, and n represents 2 to 10, preferably 2 to 4, and also preferably 2 or 4:

Therapeutic Use

The hyper-proliferative diseases, for the treatment of which the compounds according to the invention may be employed, include in particular the group of cancer and tumour diseases. In the context of the present invention, these are understood to mean especially the following diseases, but without any limitation thereto: mammary carcinomas and mammary tumours (mammary carcinomas including ductal and lobular forms, also in situ), tumours of the respiratory tract (small-cell and non-small cell carcinoma, bronchial carcinoma), cerebral tumours (e.g. of the brain stem and of the hypothalamus, astrocytoma, ependymoma, glioblastoma, glioma, medulloblastoma, meningioma and neuro-ectormal and pineal tumours), tumours of the digestive organs (carcinomas of the oesophagus, stomach, gall bladder, small intestine, large intestine, rectum and anal carcinomas), liver tumours (inter alia hepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma), tumours of the head and neck region (larynx, hypopharynx, nasopharynx, oropharynx, lips and oral cavity carcinomas, oral melanomas), skin tumours (basaliomas, spinaliomas, squamous cell carcinomas, Kaposi's sarcoma, malignant melanoma, non-melanomatous skin cancer, Merkel cell skin cancer, mast cell tumours), tumours of soft tissue (inter alia soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, chondrosarcomas, fibrosarcomas, hemangiosarcomas, leiomyosarcomas, liposarcomas, lymphosarcomas and rhabdomyosarcomas), tumours of the eyes (inter alia intraocular melanoma and retinoblastoma), tumours of the endocrine and exocrine glands (e.g. of the thyroid and parathyroid glands, pancreas and salivary gland carcinomas, adenocarcinomas), tumours of the urinary tract (tumours of the bladder, penis, kidney, renal pelvis and ureter) and tumours of the reproductive organs (carcinomas of the endometrium, cervix, ovary, vagina, vulva and uterus in women and carcinomas of the prostate and testes in men). These also include proliferative blood diseases of the blood, the lymph system and the spinal cord, in solid form and as circulating cells, such as leukaemias, lymphomas and myeloproliferative diseases, for example acute myeloid, acute lymphoblastic, chronic lymphocytic, chronic myelogenous and hairy cell leukaemia, and AIDS-correlated lymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, cutaneous T cell lymphomas, Burkitt's lymphomas and lymphomas in the central nervous system.

These well-characterized diseases in humans can also occur with a comparable aetiology in other mammals and can likewise be treated there with the compounds of the present invention.

The treatment of the cancer diseases mentioned above with the compounds according to the invention comprises both a treatment of the solid tumors and a treatment of metastasizing or circulating forms thereof.

In the context of this invention, the term “treatment” or “treat” is used in the conventional sense and means attending to, caring for and nursing a patient with the aim of combating, reducing, attenuating or alleviating a disease or health abnormality, and improving the living conditions impaired by this disease, as, for example, in the event of a cancer.

The present invention thus further provides for the use of the compounds according to the invention for the treatment and/or prevention of disorders, in particular the disorders mentioned above.

The present invention further provides for the use of the compounds according to the invention for producing a medicament for the treatment and/or prevention of disorders, in particular the disorders mentioned above.

The present invention further provides for the use of the compounds according to the invention in a method for treatment and/or prevention of disorders, in particular the disorders mentioned above.

The present invention further provides a method for treatment and/or prevention of disorders, in particular the disorders mentioned above, using an effective amount of at least one of the compounds according to the invention.

The compounds according to the invention can be used alone or, if required, in combination with one or more other pharmacologically active substances, provided that this combination does not lead to undesirable and unacceptable side effects. The present invention furthermore therefore provides medicaments containing at least one of the compounds according to the invention and one or more further active compounds, in particular for treatment and/or prevention of the abovementioned disorders.

For example, the compounds of the present invention can be combined with known anti-hyper-proliferative, cytostatic or cytotoxic substances for the treatment of cancer diseases. Examples of suitable combination active compounds include:

131I-chTNT, abarelix, abiraterone, aclarubicin, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, Alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, Hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, axitinib, azacitidine, basiliximab, belotecan, bendamustine, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calcium folinate, calcium levofolinate, capecitabine, capromab, carboplatin, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, copanlisib, crisantaspase, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin+estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, eltrombopag, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, lanreotide, lapatinib, Iasocholine, lenalidomide, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin, naloxone+pentazocine, naltrexone, nartograstim, nedaplatin, nelarabine, neridronic acid, nivolumabpentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone+sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, romidepsin, romiplostim, romurtide, roniciclib, samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur, tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine+tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.

In addition, the compounds of the present invention can be combined, for example, with binders which, by way of example, can bind to the following targets: OX-40, CD137/4-1BB, DR3, IDO1/IDO2, LAG-3, CD40.

In addition, the compounds according to the invention can also be used in combination with radiotherapy and/or surgical intervention.

Generally, the following aims can be pursued with the combination of compounds of the present invention with other cytostatically or cytotoxically active agents:

-   -   improved efficacy in slowing the growth of a tumour, in reducing         its size or even in the complete elimination thereof, compared         with treatment with an individual active compound;     -   the possibility of using the chemotherapeutics used in a lower         dosage than in the case of monotherapy;     -   the possibility of a more tolerable therapy with fewer side         effects compared with individual administration;     -   the possibility of treatment of a broader spectrum of tumour         diseases;     -   the achievement of a higher rate of response to the therapy;     -   a longer survival time of the patient compared with present-day         standard therapy.

In addition, the compounds according to the invention can also be used in combination with radiotherapy and/or surgical intervention.

The present invention further provides medicaments which comprise at least one compound according to the invention, typically together with one or more inert, nontoxic, pharmaceutically suitable excipients, and the use thereof for the aforementioned purposes.

The compounds according to the invention can act systemically and/or locally. For this purpose, they can be administered in a suitable manner, for example parenterally, possibly inhalatively or as implants or stents.

The compounds according to the invention can be administered in suitable administration forms for these administration routes.

Parenteral administration can bypass an absorption step (for example intravenously, intraarterially, intracardially, intraspinally or intralumbally) or include an absorption (for example intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). Administration forms suitable for parenteral administration include preparations for injection and infusion in the form of solutions, suspensions, emulsions or lyophilizates. Preference is given to parenteral administration, especially intravenous administration.

In general, it has been found to be advantageous in the case of parenteral administration to administer amounts of from about 0.001 to 1 mg/kg, preferably about 0.01 to 0.5 mg/kg, of body weight to achieve effective results.

It may nevertheless be necessary where appropriate to deviate from the stated amounts, specifically as a function of body weight, route of administration, individual response to the active compound, nature of the preparation and time or interval over which administration takes place. Thus, in some cases less than the abovementioned minimum amount may be sufficient, while in other cases the upper limit mentioned must be exceeded. In the case of administration of greater amounts, it may be advisable to divide them into several individual doses over the day.

EXAMPLES

The examples which follow illustrate the invention. The invention is not restricted to the examples.

Unless stated otherwise, the percentages in the tests and examples which follow are percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for the liquid/liquid solutions are in each case based on volume.

Synthesis Routes:

Examplary for the working examples, the schemes below show exemplary synthesis routes leading to the working examples:

with n is 2 or 4.

-   -   a. HATU, DIPEA, DMF, rt; b. ACN, Buffer pH8, rt; c. HCl 4M,         dioxane, rt

with n is 2 or 4.

[a): for example benzyl bromide, Cs₂CO₃, DMF, RT; b) for example Pd(dppf)₂Cl₂, DMF, NaCO₃, 85° C.; c) for example LiAlH₄, THF, 0° C.; MnO₂, DCM, RT; d) for example Ti(iOPr)₄, THF, RT; e) for example tBuLi, THF, −78° C.; MeOH, NH₄Cl; f) for example HCl/1,4-dioxane]

-   -   a. HATU, DIPEA, DMF, rt; b) Pd/C 10%, MeOH; c) HATU, DIPEA, DMF,         rt, d) Pd/C 10%, MeOH

[a): for example sodium triacetoxyborohydride, acetic acid, DCM, RT; b) for example acetoxyacetyl chloride, NEt₃, DCM, RT; c) for example LiOH, THF/water, RT; d) for example H₂, Pd—C, EtOH, RT; e) for example Teoc-OSu, NEt₃, dioxane, RT; f) for example Fmoc-Cl, diisopropylethylamine, dioxane/water 2:1, RT]

[a) HATU, DMF, diisopropylethylamine, RT; b) TFA, DCM, RT; c) HATU, DMF, diisopropylethylamine, RT; d) zinc chloride, trifluorethanol, 50° C.; e) H₂, Pd/C, THF/water, RT]

[a) HATU, DMF, diisopropylethylamine, RT; b) H₂, Pd/C, THF/water, RT; c) zinc chloride, trifluorethanol, 50° C.]

with n is 2 or 4.

[a) sodium triacetoxyborohydride, acetic acid, DCM, RT; b) acetoxyacetylchloride, triethylamine, DCM, RT; c) L-Cystein, NaHCO₃, DBU, isopropanol/water, RT; d) Boc-6-aminocaproic acid, HATU, DMF, Diisopropylethylamin, RT; e) Zinkchlorid, Trifluorethanol, 50° C., EDTA.]

A. Examples Abbreviations and Acronyms

-   A431NS human tumour cell line -   A549 human tumour cell line -   ABCB1 ATP-binding cassette sub-family B member 1 (synonym for P-gp     and MDR1) -   abs. absolute -   Ac acetyl -   ACN acetonitrile -   aq. aqueous, aqueous solution -   ATP adenosine triphosphate -   BCRP breast cancer resistance protein, an efflux transporter -   BEP 2-bromo-1-ethylpyridinium tetrafluoroborate -   Boc tert-butoxycarbonyl -   br. broad (in NMR) -   Ex. Example -   CI chemical ionization (in MS) -   D doublet (in NMR) -   D day(s) -   TLC thin-layer chromatography -   DCI direct chemical ionization (in MS) -   Dd doublet of doublets (in NMR) -   DMAP 4-N,N-dimethylaminopyridine -   DME 1,2-dimethoxyethane -   DMEM Dulbecco's Modified Eagle Medium (standardized nutrient medium     for cell culture) -   DMF N,N-dimethylformamide -   DMSO dimethyl sulphoxide -   DPBS, D-PBS, PBS Dulbecco's phosphate-buffered salt solution     -   PBS=DPBS=D-PBS, pH 7.4, from Sigma, No D8537 composition:     -   0.2 g KCl     -   0.2 g KH₂PO₄ (anhyd)     -   8.0 g NaCl     -   1.15 g Na₂HPO₄ (anhyd)     -   made up ad 1 l with H₂O -   Dt doublet of triplets (in NMR) -   DTT DL-dithiothreitol -   EDC N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride -   EGFR epidermal growth factor receptor -   EI electron impact ionization (in MS) -   ELISA enzyme-linked immunosorbent assay -   eq. equivalent(s) -   ESI electrospray ionization (in MS) -   ESI-MicroTofq ESI-MicroTofq (name of the mass spectrometer with     Tof=time of flight and q=quadrupol) -   FCS foetal calf serum -   Fmoc (9H-fluoren-9-ylmethoxy)carbonyl -   GTP guanosine-5′-triphosphate -   H hour(s) -   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium     hexafluorophosphate -   HCT-116 human tumour cell line -   HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid -   HOAc acetic acid -   HOAt 1-hydroxy-7-azabenzotriazole -   HOBt 1-hydroxy-1H-benzotriazole hydrate -   HOSu N-hydroxysuccinimide -   HPLC high-pressure high-performance liquid chromatography -   HT29 human tumour cell line -   IC₅₀ half-maximal inhibitory concentration -   i.m. intramuscularly, administration into the muscle -   i.v. intravenously, administration into the vein -   conc. concentrated -   LC-MS liquid chromatography-coupled mass spectroscopy -   LLC-PK1 cells Lewis lung carcinoma pork kidney cell line -   L-MDR human MDR1 transfected LLC-PK1 cells -   M multiplet (in NMR) -   MDR1 multidrug resistance protein 1 -   MeCN acetonitrile -   Me methyl -   Min minute(s) -   MS mass spectrometry -   MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide -   NCI-H292 human tumour cell line -   NCI-H520 human tumour cell line -   NMM N-methylmorpholine -   NMP N-methyl-2-pyrrolidinone -   NMR nuclear magnetic resonance spectrometry -   NMRI mouse strain, originates from the Naval Medical Research     Institute (NMRI) -   NSCLC non small cell lung cancer -   PBS phosphate-buffered salt solution -   Pd/C palladium on activated carbon -   P-gp P-gycoprotein, a transporter protein -   PNGaseF enzyme for cleaving sugar -   quant. quantitative (in yield) -   Quart quartet (in NMR) -   Quint quintet (in NMR) -   R_(f) retention index (in TLC) -   RT room temperature -   R_(t) retention time (in HPLC) -   S singlet (in NMR) -   s.c. subcutaneously, administration under the skin -   SCC-4 human tumour cell line -   SCC-9 human tumour cell line -   SCID mice test mice with severe combined immunodeficiency -   t triplet (in NMR) -   TBAF tetra-n-butylammonium fluoride -   TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl -   tert. tertiary -   TFA trifluoroacetic acid -   THF tetrahydrofuran -   T3P® 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide -   UV ultraviolet spectrometry -   v/v ratio by volume (of a solution) -   Z benzyloxycarbonyl     Amino Acid Abbreviations -   Ala=Alanine -   Arg=Arginine -   Asn=Asparagine -   Asp=Aspartic acid -   Cys=Cysteine -   Glu=Glutamic acid -   Gln=Glutamine -   Gly=Glycine -   His=Histidine -   Ile=Isoleucine -   Leu=Leucine -   Lys=Lysine -   Met=Methionine -   Nva=Norvaline -   Phe=Phenylalanine -   Pro=Proline -   Ser=Serine -   Thr=Threonine -   Trp=Tryptophan -   Tyr=Tyrosine -   Val=Valine

If, in the context of the present disclosure, no temperature is given in the description of a reaction, room temperature should always be assumed.

HPLC and LC-MS Methods:

Method 1 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8μ 50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid; mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% A oven: 50° C.; flow rate: 0.40 ml/min; UV detection: 208-400 nm.

Method 2 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, BEH300, 2.1×150 mm, C18 1.7 μm; mobile phase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l of acetonitrile+0.01% formic acid; gradient: 0.0 min 2% B→1.5 min 2% B→8.5 min 95% B→10.0 min 95% B; oven: 50° C.; flow rate: 0.50 ml/min; UV detection: 220 nm

Method 3 (LC-MS):

MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100 series; column: Agilent ZORBAX Extend-C18 3.0×50 mm 3.5 micron; mobile phase A: 1 l of water+0.01 mol of ammonium carbonate, mobile phase B: 1 l of acetonitrile; gradient: 0.0 min 98% A→0.2 min 98% A→3.0 min 5% A→4.5 min 5% A; oven: 40° C.; flow rate: 1.75 ml/min; UV detection: 210 nm

Method 4 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 μm; mobile phase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l of acetonitrile+0.01% formic acid; gradient: 0.0 min 10% B→0.3 min 10% B→1.7 min 95% B→2.5 min 95% B; oven: 50° C.; flow rate: 1.20 ml/min; UV detection: 210 nm

Method 5 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC system; column: Waters Acquity UPLC HSS T3 1.8μ 50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid; mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% A oven: 50° C.; flow rate: 0.35 ml/min; UV detection: 210-400 nm.

Method 6 (LC-MS):

Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column: Thermo Hypersil GOLD 1.9μ 50×1 mm; mobile phase A: 1 l of water+0.5 ml of 50% strength formic acid; mobile phase B: 1 l of acetonitrile+0.5 ml of 50% strength formic acid; gradient: 0.0 min 97% A→0.5 min 97% A→3.2 min 5% A→4.0 min 5% A; oven: 50° C.; flow rate: 0.3 ml/min; UV detection: 210 nm.

Method 7 (LC-MS):

Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; column: Waters Acquity UPLC HSS T3 1.8μ 50×2.1 mm; mobile phase A: 1 l of water+0.25 ml of 99% strength formic acid; mobile phase B: 1 l of acetonitrile+0.25 ml of 99% strength formic acid; gradient: 0.0 min 90% A→0.3 min 90% A→1.7 min 5% A→3.0 min 5% A; oven: 50° C.; flow rate: 1.20 ml/min; UV detection: 205-305 nm.

Method 8 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: Waters Acquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 μm; mobile phase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l of acetonitrile+0.01% formic acid; gradient: 0.0 min 2% B→2.0 min 2% B→13.0 min 90% B→15.0 min 90% B; oven: 50° C.; flow rate: 1.20 ml/min; UV detection: 210 nm

Method 9: LC-MS-Prep Purification Method for Examples 181-191 (Method LIND-LC-MS-Prep)

MS instrument: Waters; HPLC instrument: Waters (column Waters X-Bridge C18, 19 mm×50 mm, 5 μm, mobile phase A: water+0.05% ammonia, mobile phase B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection: DAD; 210-400 nm).

or:

MS instrument: Waters; HPLC instrument: Waters (column Phenomenex Luna 5μ C18(2) 100A, AXIA Tech. 50×21.2 mm, mobile phase A: water+0.05% formic acid, mobile phase B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UV detection: DAD; 210-400 nm).

Method 10: LC-MS Analysis Method for Examples 181-191 (LIND_SQD_SB_AQ)

MS instrument: Waters SQD; HPLC instrument: Waters UPLC; column: Zorbax SB-Aq (Agilent), 50 mm×2.1 mm, 1.8 μm; mobile phase A: water+0.025% formic acid, mobile phase B: acetonitrile (ULC)+0.025% formic acid; gradient: 0.0 min 98% A-0.9 min 25% A-1.0 min 5% A-1.4 min 5% A-1.41 min 98% A-1.5 min 98% A; oven: 40° C.; flow rate: 0.600 ml/min; UV detection: DAD; 210 nm.

Method 11 (HPLC):

-   Instrument: HP1100 Series -   Column: Merck Chromolith SpeedROD RP-18e, 50-4.6 mm, Cat. No.     1.51450.0001, precolumn Chromolith Guard Cartridge Kit, RP-18e,     5-4.6 mm, Cat. No. 1.51470.0001 -   Gradient: flow rate 5 ml/min     -   injection volume 5 μl     -   solvent A: HClO₄ (70% strength) in water (4 ml/l)     -   solvent B: acetonitrile     -   start 20% B     -   0.50 min 20% B     -   3.00 min 90% B     -   3.50 min 90% B     -   3.51 min 20% B     -   4.00 min 20% B     -   column temperature: 40° C.         Wavelength: 210 nm

All reactants or reagents whose preparation is not described explicitly hereinafter were purchased commercially from generally accessible sources. For all other reactants or reagents whose preparation likewise is not described hereinafter and which were not commercially obtainable or were obtained from sources which are not generally accessible, a reference is given to the published literature in which their preparation is described.

Method 12 (LC-MS):

Instrument MS: Thermo Scientific FT-MS; Instrument UHPLC+: Thermo Scientific UltiMate 3000; Säule: Waters, HSST3, 2.1×75 mm, C18 1.8 μm; Eluent A: 1 l Wasser+0.01% Ameisensäure; Eluent B: 1 l Acetonitril+0.01% Ameisensaure; Gradient: 0.0 min 10% B→2.5 min 95% B→3.5 min 95% B; Ofen: 50° C.; Fluss: 0.90 ml/min; UV-Detektion: 210 nm/Optimum Integration Path 210-300 nm

Method 13: (LC-MS):

Instrument MS: Waters (Micromass) Quattro Micro; Instrument Waters UPLC Acquity; Säule: Waters BEH C18 1.7μ 50×2.1 mm; Eluent A: 1 l Wasser+0.01 mol Ammoniumformiat, Eluent B: 1 l Acetonitril; Gradient: 0.0 min 95% A→0.1 min 95% A→2.0 min 15% A→2.5 min 15% A→2.51 min 10% A→3.0 min 10% A; Ofen: 40° C.; Fluss: 0.5 ml/min; UV-Detektion: 210 nm

Starting Materials and Intermediates Intermediate C1 Trifluoroacetic acid-(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropan-1-amine (1:1)

The title compound was prepared as described in WO2006/002326.

Intermediate C2 tert-Butyl(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino)-2-[(tert-butoxycarbonyl)amino]butanoate

4.22 g (14.5 mmol) of tert-butyl N-(tert-butoxycarbonyl)-L-homoserinate were dissolved in 180 ml of dichloromethane, and 3.5 ml of pyridine and 9.2 g (21.7 mmol) of 1,1,1-triacetoxy-llambda⁵,2-benziodoxol-3(1H)-one were then added. The reaction was stirred at RT for 1 h and then diluted with 500 ml of dichloromethane and extracted twice with 10% strength sodium thiosulphate solution and then extracted successively twice with 5% strength citric acid and twice with 10% strength sodium bicarbonate solution. The organic phase was separated off, dried over magnesium sulphate and then dried under reduced pressure. The residue was taken up in diethyl ether, and HCl (solution in diethyl ether) was added. The precipitate was filtered off and the filtrate was then concentrated and lyophilized from acetonitrile/water. This gave 3.7 g (93%) of tert-butyl (2S)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoate which were used without further purification for the next step. (R_(f): 0.5 (DCM/methanol 95/5).

3.5 g (9.85 mmol) of Intermediate C1 were dissolved in 160 ml of DCM, and 3.13 g (14.77 mmol) of sodium triacetoxyborohydride and 0.7 ml of acetic acid were added. After 5 min of stirring at RT, 3.23 g (11.85 mmol) of tert-butyl (2S)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoate were added and the reaction was stirred at RT for a further 30 min. The solvent was then evaporated under reduced pressure and the residue was taken up in acetonitrile/water. The precipitated solid was filtered off and dried, giving 5.46 g (84%) of the title compound.

HPLC (Method 11): R_(t)=2.5 min;

LC-MS (Method 1): R_(t)=1.13 min; MS (ESIpos): m/z=613 (M+H)⁺.

Intermediate C3 (2S)-4-[(Acetoxyacetyl){(1R)-1-[1benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid

5.46 g (8.24 mmol) of Intermediate C2 were dissolved in 160 ml of DCM, and 4.8 ml of triethylamine and 2.2 ml (20.6 mmol) of acetoxyacetyl chloride then were added. The reaction was stirred at RT overnight and then concentrated under reduced pressure. The residue was taken up in ethyl acetate and extracted three times with saturated sodium bicarbonate solution and then with saturated sodium chloride solution. The organic phase was dried over sodium sulphate and then concentrated. The residue was purified by column chromatography on Biotage/Isolera (SNAP 340 g) using the mobile phase cyclohexane/ethyl acetate 2:1. This gave 4.57 g (75%) of the acylated intermediate.

LC-MS (Method 1): R_(t)=1.49 min; MS (ESIpos): m/z=713 (M+H)⁺.

1 g (1.36 mmol) of this intermediate was dissolved in 20 ml of DCM, and 20 ml of TFA were added. After 5 h of stirring at RT, the mixture was concentrated and the residue was triturated twice with n-pentane. In each case, the n-pentane was decanted off and the solid that remained was dried under high vacuum. This gave 1.1 g of (2S)-4-[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}amino]-2-aminobutanoic acid/trifluoroacetic acid (1:1). LC-MS (Method 1): R_(t)=0.93 min; MS (ESIpos): m/z=557 (M+H)⁺.

0.91 g (1.57 mmol) of this intermediate were dissolved in 70 ml of DCM, and 3.43 g (15.7 mmol) of di-tert-butyl dicarbonate and 4.1 ml of N,N-diisopropylethylamine were added. After 30 min of stirring at RT, the reaction was diluted with DCM and extracted with 5% strength citric acid. The organic phase was dried over sodium sulphate and concentrated. The residue was triturated twice with n-pentane and in each case the n-pentane was decanted off. The solid that remained was lyophilized from acetonitrile/water 1:1, giving 1.11 g of the title compound.

HPLC (Method 11): R_(t)=2.55 min;

LC-MS (Method 1): R_(t)=1.3 min; MS (ESIpos): m/z=657 (M+H)⁺.

Intermediate C4 (2S)-2-Amino-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoic acid/trifluoroacetic acid (1:1)

5.46 g (8.24 mmol) of Intermediate C2 were dissolved in 160 ml of DCM, and 4.8 ml of triethylamine and 2.2 ml (20.6 mmol) of acetoxyacetyl chloride were added. The reaction was stirred at RT overnight and then concentrated under reduced pressure. The residue was taken up in ethyl acetate and extracted three times with saturated sodium bicarbonate solution and then with saturated sodium chloride solution. The organic phase was dried over sodium sulphate and then concentrated. The residue was purified by column chromatography on Biotage/Isolera (SNAP 340 g) using the mobile phase cyclohexane/ethyl acetate 2:1. This gave 4.57 g (75%) of the acylated intermediate.

LC-MS (Method 1): R_(t)=1.49 min; MS (ESIpos): m/z=713 (M+H)⁺.

1.5 g (2.035 mmol) of this intermediate were taken up in 50 ml of ethanol, and 5.8 ml of a 40% strength solution of methanamine in water was added. The reaction was stirred at 50° C. for 4 h and then concentrated. The residue was taken up in DCM and washed twice with water. The organic phase was dried over magnesium sulphate and then concentrated.

The residue was dried under high vacuum. This gave 1.235 mg of this intermediate, which were reacted further without further purification.

1.235 mg (1.5 mmol) of this intermediate were dissolved in 15 ml of DCM, and 15 ml of TFA were added. After 4 h of stirring at RT, the mixture was concentrated. The residue was purified by preparative HPLC. The appropriate fractions were concentrated and the residue was lyophilized from acetonitrile. This gave 1.04 g (quant) of the title compound.

HPLC (Method 11): R_(t)=1.9 min;

LC-MS (Method 1): R_(t)=0.89 min; MS (ESIpos): m/z=515 (M+H)⁺.

Intermediate C5 (2S)-4-[{(1R)-1-[1Benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid

0.9 g (1.24 mmol) of Intermediate C4 was dissolved in 60 ml of DCM, and 2.7 g (12.5 mmol) of di-tert-butyl dicarbonate and 3.3 ml of N,N-diisopropylethylamine were added. After 45 min of stirring at RT, the reaction was concentrated and the residue was taken up in diethyl ether, and n-pentane was added until the mixture started to get cloudy. The reaction was cooled to 0° C. and then decanted. Once more, n-pentane was added to the residue and the mixture was decanted. The solid that remained was lyophilized from acetonitrile/water 1:1, giving 0.95 g (quant) of the title compound.

HPLC (Method 11): R_(t)=2.5 min;

LC-MS (Method 1): R_(t)=1.27 min; MS (ESIpos): m/z=615 (M+H)⁺.

Intermediate C52 (1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine

10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate were initially charged in 100.0 ml of DMF, and 20.76 g (63.72 mmol) of caesium carbonate and 9.22 g (53.91 mmol) of benzyl bromide were added. The reaction mixture was stirred at RT overnight. The reaction mixture was partitioned between water and ethyl acetate and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over magnesium sulphate and the solvent was evaporated under reduced pressure. The reaction was repeated with 90.0 g of methyl 4-bromo-1H-pyrrole-2-carboxylate.

The two combined reactions were purified by preparative RP-HPLC (column: Daiso 300×100; 10μ, flow rate: 250 ml/min, MeCN/water). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 125.15 g (87% of theory) of the compound methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate.

LC-MS (Method 1): R_(t)=1.18 min; MS (ESIpos): m/z=295 [M+H]⁺.

Under argon, 4.80 g (16.32 mmol) of methyl 1-benzyl-4-bromo-1H-pyrrole-2-carboxylate were initially charged in DMF, and 3.61 g (22.85 mmol) of (2,5-difluorophenyl)boronic acid, 19.20 ml of saturated sodium carbonate solution and 1.33 g (1.63 mmol) of [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II):dichloromethane were added. The reaction mixture was stirred at 85° C. overnight. The reaction mixture was filtered through Celite and the filter cake was washed with ethyl acetate. The organic phase was extracted with water and then washed with saturated NaCl solution. The organic phase was dried over magnesium sulphate and the solvent was evaporated under reduced pressure. The residue was purified on silica gel (mobile phase: cyclohexane/ethyl acetate 100:3). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 3.60 g (67% of theory) of the compound methyl 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate.

LC-MS (Method 7): R_(t)=1.59 min; MS (ESIpos): m/z=328 [M+H]⁺.

3.60 g (11.00 mmol) of methyl 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate were initially charged in 90.0 ml of THF, and 1.04 g (27.50 mmol) of lithium aluminium hydride (2.4 M in THF) were added at 0° C. The reaction mixture was stirred at 0° C. for 30 minutes. At 0° C., saturated potassium sodium tartrate solution was added, and ethyl acetate was added to the reaction mixture. The organic phase was extracted three times with saturated potassium sodium tartrate solution. The organic phase was washed once with saturated NaCl solution and dried over magnesium sulphate. The solvent was evaporated under reduced pressure and the residue was dissolved in 30.0 ml of dichloromethane. 3.38 g (32.99 mmol) of manganese(IV) oxide were added and the mixture was stirred at RT for 48 h. Another 2.20 g (21.47 mmol) of manganese(IV) oxide were added and the mixture was stirred at RT overnight. The reaction mixture was filtered through Celite and the filter cake was washed with dichloromethane. The solvent was evaporated under reduced pressure and the residue 2.80 g of (1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde) was used without further purification in the next step of the synthesis.

LC-MS (Method 7): R_(t)=1.48 min; MS (ESIpos): m/z=298 [M+H]⁺.

28.21 g (94.88 mmol) of 1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde together with 23.00 g (189.77 mmol) of (R)-2-methylpropane-2-sulphinamide were initially charged in 403.0 ml of absolute THF, and 7.42 g (237.21 mmol) of titanium(IV) isopropoxide were added and the mixture was stirred at RT overnight. 500.0 ml of saturated NaCl solution and 1000.0 ml of ethyl acetate were added, and the mixture was stirred at RT for 1 h. The mixture was filtered through kieselguhr and the filtrate was washed twice with saturated NaCl solution. The organic phase was dried over magnesium sulphate, the solvent was evaporated under reduced pressure and the residue was purified using Biotage Isolera (silica gel, column 1500+340 g SNAP, flow rate 200 ml/min, ethyl acetate/cyclohexane 1:10).

LC-MS (Method 7): R_(t)=1.63 min; MS (ESIpos): m/z=401 [M+H]⁺.

25.00 g (62.42 mmol) of (R)—N—{(E/Z)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]methylene}-2-methylpropane-2-sulphinamide were initially charged in absolute THF under argon and cooled to −78° C. 12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane) were then added at −78° C. and the mixture was stirred at this temperature for 3 h. At −78° C., 71.4 ml of methanol and 214.3 ml of saturated ammonium chloride solution were then added in succession, and the reaction mixture was allowed to warm to RT and stirred at RT for 1 h. The mixture was diluted with ethyl acetate and washed with water. The organic phase was dried over magnesium sulphate and the solvent was evaporated under reduced pressure. The residue (R)—N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulphinamide was used without further purification in the next step of the synthesis.

LC-MS (Method 6): R_(t)=2.97 min; MS (ESIpos): m/z=459 [M+H]⁺.

28.00 g (61.05 mmol) of (R)—N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulphinamide were initially charged in 186.7 ml of 1,4-dioxane, and 45.8 ml of HCl in 1,4-dioxane solution (4.0 M) were then added. The reaction mixture was stirred at RT for 2 h and the solvent was evaporated under reduced pressure. The residue was purified by preparative RP-HPLC (column: (column: Kinetix 100×30; flow rate: 60 ml/min, MeCN/water). The acetonitrile was evaporated under reduced pressure and dichloromethane was added to the aqueous residue. The organic phase was washed with sodium bicarbonate solution and dried over magnesium sulphate. The solvent was evaporated under reduced pressure and the residue was dried under high vacuum. This gave 16.2 g (75% of theory) of the title compound.

LC-MS (Method 6): R_(t)=2.10 min; MS (ESIpos): m/z=338 [M-NH₂]⁺, 709 [2M+H]⁺.

¹H NMR (400 MHz, DMSO-d₆): δ [ppm]=0.87 (s, 9H), 1.53 (s, 2H), 3.59 (s, 1H), 5.24 (d, 2H), 6.56 (s, 1H), 6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m, 1H), 7.26 (m, 2H), 7.34 (m, 2H), 7.46 (m, 1H).

Intermediate C53 (2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic acid

First, Intermediate C52 was reductively alkylated with benzyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate analogously to C2. The secondary amino group was then acylated with 2-chloro-2-oxoethyl acetate and the two ester groups were then hydrolysed with 2M lithium hydroxide solution in methanol. The intermediate obtained in this manner was dissolved in ethanol, palladium on carbon (10%) was added and the mixture was hydrogenated at RT with hydrogen under standard pressure for 1 h. The deprotected compound was taken up in dioxane/water 2:1 and in the last step the Fmoc protective group was introduced using 9H-fluoren-9-ylmethyl chlorocarbonate in the presence of N,N-diisopropylethylamine.

LC-MS (Method 1): R_(t)=1.37 min; MS (ESIpos): m/z=734 (M−H)⁻.

Intermediate C58 (2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoic acid

First, Intermediate C52 was reductively alkylated with benzyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate analogously to Intermediate C2. First, Intermediate C52 was reductively alkylated with benzyl (2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate analogously to C2. The secondary amino group was then acylated with 2-chloro-2-oxoethyl acetate as described for Intermediate C27, and the two ester groups were then hydrolysed with 2M lithium hydroxide solution in methanol. The intermediate obtained in this manner was dissolved in ethanol, palladium on carbon (10%) was added and the mixture was hydrogenated at RT with hydrogen under standard pressure for 1 h.

500 mg (0.886 mmol) of this fully deprotected intermediate were taken up in 60 ml of dioxane, and 253 mg (0.975 mmol) of 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione and 198 μl of triethylamine were added. After 24 h of stirring at RT, the reaction was concentrated and the residue was purified by preparative HPLC. Combination of the appropriate fractions, concentration under reduced pressure and drying under high vacuum gave 312 mg (50% of theory) of the title compound.

LC-MS (Method 5): R_(t)=4.61 min; MS (ESIpos): m/z=658 (M+H)⁻.

Intermediate C61 N-[(2S)-4-[{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-beta-alanine

The title compound was prepared by coupling 60 mg (0.091 mmol) of Intermediate C58 with methyl B-alaninate, followed by ester cleavage with 2M lithium hydroxide solution. This gave 67 mg (61% of theory) of the title compound over 2 steps.

LC-MS (Method 1): R_(t)=1.29 min; MS (ESIpos): m/z=729 (M+H)⁺.

Intermediate C69 11-{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-14-thia-7,11-diaza-2-silaheptadecan-17-oic acid

117.0 mg (0.19 mmol) of (2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) and 21.6 mg (0.20 mmol) of 3-sulphanylpropanoic acid were initially charged in 3.0 ml of methanol, 89.5 mg (0.65 mmol) of potassium carbonate were added and the mixture was stirred at 50° C. for 4 h. The reaction mixture was diluted with ethyl acetate and the organic phase was washed with water and saturated NaCl solution. The organic phase was dried over magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was dried under high vacuum. The residue was used without further purification in the next step of the synthesis. This gave 106.1 mg (73% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.42 min; MS (ESIneg): m/z=700 (M−H)⁻.

Intermediate C70 (2-(Trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate

990.0 mg (2.79 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine (intermediate C52) were initially charged in 15.0 ml of dichloromethane, and 828.8 mg (3.91 mmol) of sodium triacetoxyborohydride and 129.9 mg (3.21 mmol) of acetic acid were added, and the mixture was stirred at RT for 5 min. 698.1 mg (3.21 mmol) of 2-(trimethylsilyl)ethyl (3-oxopropyl)carbamate (Intermediate L15) dissolved in 15.0 ml of dichloromethane were added, and the reaction mixture was stirred at RT overnight. The reaction mixture was diluted with ethyl acetate and the organic phase was washed in each case twice with saturated sodium carbonate solution and saturated NaCl solution. The organic phase was dried over magnesium sulphate and the solvent was evaporated under reduced pressure. The residue was purified using silica gel (mobile phase: dichloromethane/methanol 100:2). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 1.25 g (73% of theory) of the compound 2-(trimethylsilyl)ethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate

LC-MS (Method 1): R_(t)=1.09 min; MS (ESIpos): m/z=556 (M+H)⁺.

908.1 mg (1.63 mmol) of 2-(trimethylsilyl)ethyl [3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate and 545.6 mg (5.39 mmol) of triethylamine were initially charged in 10.0 ml of dichloromethane, and the mixture was cooled to 0° C. At this temperature, 590.5 mg (5.23 mmol) of chloroacetyl chloride were added and the mixture was stirred at RT overnight. The reaction mixture was diluted with ethyl acetate and the organic phase was washed in each case three times with saturated sodium bicarbonate solution and saturated ammonium chloride solution. The organic phase was washed with saturated NaCl solution and dried over magnesium sulphate. The residue was purified by preparative RP-HPLC (column: Reprosil 250×30; 10μ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 673.8 mg (65% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.53 min; MS (ESIneg): m/z=676 (M+HCOO⁻)⁻.

Intermediate C71 S-(11-{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-L-cysteine/trifluoroacetic acid (1:1)

536.6 mg (4.43 mmol) of L-cysteine were suspended in 2.5 ml of water together with 531.5 mg (6.33 mmol) of sodium bicarbonate. 400.0 mg (0.63 mmol) of 2-(trimethylsilyl)ethyl {3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate (Intermediate C70) dissolved in 25.0 ml of isopropanol and 1.16 g (7.59 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene were added. The reaction mixture was stirred at 50° C. for 1.5 h. Ethyl acetate was added to the reaction mixture and the organic phase was washed repeatedly with saturated sodium bicarbonate solution and once with sat. NaCl solution. The organic phase was dried over magnesium sulphate, the solvent was evaporated under reduced pressure and the residue was dried under high vacuum. The residue was purified by preparative RP-HPLC (column: Reprosil 250×30; 10μ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 449.5 mg (86% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.20 min; MS (ESIpos): m/z=717 (M+H)⁺.

Intermediate C74 2-(Trimethylsilyl)ethyl 3-amino-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoyl]-D-alaninate trifluoroacetate (1:1)

75 mg (0.114 mmol) of intermediate C58 were dissolved in 12.5 ml DMF and coupled with 78 mg (0.171 mmol) of intermediate L6 in the presence of 65 mg (0.11 mmol) HATU and 79 μL N,N-diisopropylethylamine. After purification via preparative HPLC, the residue was dissolved in 20 mL ethanol and hydrogenated for 1 h in presence of 10% Pd/C at room temperature at atmospheric pressure. After filtration of the catalyst, the solvent was evaporated under reduced pressure. The residue was purified over preparative HPLC and lyophilized to afford 63 mg (64% over 2 steps) of the title compound.

LC-MS (Method 1): R_(t)=1.16 min; MS (EIpos): m/z=844 [M+H]⁺.

Intermediate C75 Methyl (2S)-4-[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethyl propyl}amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate

To a solution of 4.3 g (12.2 mmol) of intermediate C52 in 525 mL DCM was added 3.63 g (17.12 mmol) sodium triacetoxyborhydride and 8.4 mL acetic acid. After stirring the mixture at room temperature for 5 min, a solution of 3.23 g (11.85 mmol) methyl-(2S)-4-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate (prepared from (3S)-3-amino-4-methoxy-4-oxobutanoic acid using classical method) in 175 mL DCM was added and the reaction mixture was stirred for 45 min at room temperature. The crude mixture was diluted with DCM and washed twice with 100 mL of a saturated sodium hydrogen carbonate solution and then with brine. The organic layer was dried over magnesium sulfate and evaporated. The residue was purified over preparative HPLC to afford 4.6 g (61%) methyl (2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate.

LC-MS (Method 12): R_(t)=1.97 min; MS (ESIpos): m/z=614.32 (M+H)⁺.

To a solution of 200 mg (0.33 mmol) methyl (2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate 10 mL DCM were added 105 μL triethylamine and 77 μL (0.717 mmol) acetoxyacetylchloride. The reaction mixture was stirred overnight at room temperature diluted with ethyl acetate and washed twice with a saturated sodium hydrogen carbonate solution and then with brine. The organic layer was dried over magnesium sulfate and evaporated to afford 213 mg (75%) of the title compound.

LC-MS (Method 1): R_(t)=1.46 min; MS (ESIpos): m/z=714 (M+H)⁺.

Intermediate C88 tert-Butyl (3R)-3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)methyl]pyrrolidine-1-carboxylate trifluoroacetate (1:1) (mixture of stereoisomers)

To a solution of 2.04 g (5.75 mmol) tert-butyl (3R)-3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)methyl]pyrrolidine-1-carboxylate trifluoroacetate (1:1) (intermediate C52) in 51 ml dichlormethane were added 1.71 g (8.05 mmol) sodium triacetoxyborhydride and 0.40 g (6.61 mmol) acetic acid and the reaction mixture was stirred at room temperature for 5 min. A solution of 1.32 g (6.61 mmol) tert-butyl 3-formylpyrrolidine-1-carboxylate in 20 ml dichlormethane was then added and the mixture was stirred overnight at room temperature. Ethyl acetate was then added and the organic phase was washed with a saturated sodium carbonate solution and brine. The organic phase was dried over magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was dried under high vacuum. The residue was used without further purification in the next step of the synthesis. This gave 1.86 g (50% of theory) of the title compound.

LC-MS (Method 1): R_(t)=0.99 min; MS (ESIpos): m/z=538 (M+H−CF₃CO₂H)⁺.

Intermediate C89 tert-Butyl (3R)-3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]methyl}pyrrolidine-1-carboxylate

To a solution of 2.89 g (4.19 mmol, 80% purity) of tert-butyl (3R)-3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)methyl]pyrrolidine-1-carboxylate (intermediate C88) in 42 ml dichlormethane with 4 Å molecular sieves were added 1.36 g (13.42 mmol) triethylamine and 2.13 g (18.87 mmol) of chloracetyl chloride. The reaction mixture was stirred at room temperature for 5 h and the solvent was then evaporated. The residue was purified over preparative HPLC to afford 449 mg (17% d. Th.) of isomere 1 and 442 mg (17% d. Th) of isomere 2 of the title compound.

Isomere 1 LC-MS (Method 1): R_(t)=2.74 min; MS (ESIpos): m/z=614 (M+H)⁺.

Isomere 2 LC-MS (Method 1): R_(t)=2.78 min; MS (ESIpos): m/z=614 (M+H)⁺.

Intermediate C90 S-[2-({(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3R)-1-(tert- butoxycarbonyl)pyrrolidin-3-yl]methyl}amino)-2-oxoethyl]-L-cysteine (Isomere 1)

To a solution of 493 mg (4.07 mmol) L-cysteine in 2.3 mL water were added 489 mg (5.82 mmol) of sodium hydrogencarbonate followed by a solution of 357 mg (0.58 mmol) of tert-butyl (3R)-3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]methyl}pyrrolidine-1-carboxylate (intermediate C89, isomere 1) in 23.0 mL iso-propanol and 1.06 g (6.98 mmol) of 1,8-diazabicyclo(5.4.0)undec-7-ene. The reaction mixture was stirred at 50° C. for 3h. Ethyl acetate was then added and the organic phase was washed with a saturated sodium carbonate solution and brine. The organic phase was dried over magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was dried under high vacuum. The residue was used without further purification in the next step of the synthesis. This gave 255 mg (62% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.09 min; MS (ESIpos): m/z=699 (M+H)⁺.

Intermediate C95 3-{[2-({(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3R)-1-(tert- butoxycarbonyl)pyrrolidin-3-yl]methyl}amino)-2-oxoethyl]sulfanyl}propanoic acid (Isomer 1)

To a mixture of 384.0 mg (0.62 mmol) of tert-butyl (3R)-3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]methyl}pyrrolidine-1-carboxylate (intermediate C89, isomere 1) and 73.0 mg (0.69 mmol) of 3-sulfanylpropanoic acid in 14 ml Methanol and one drop water was added 302.5 mg (2.19 mmol) of potassium carbonate. The reaction mixture was stirred at 50° C. for 2.5 h. Ethyl acetate was then added and the organic phase was then washed with water and brine, dried over magnesium sulfate and evaporated. under reduced pressure. The residue was dried under high vacuum and used without further purification in the next step of the synthesis. This gave 358.0 mg (84% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.33 min; MS (ESIpos): m/z=684 (M+H)⁺.

Intermediate C101 N-(3-Aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide

150.0 mg (0.42 mmol) of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine (intermediate C52) were initially charged in 2.0 ml of dichloromethane, and 29.2 mg (0.49 mmol) of HOAc and 125.6 mg (0.59 mmol) of sodium triacetoxyborohydride were added and the mixture was stirred at RT for 5 min. 98.9 mg (0.49 mmol) of 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propanal were added. The reaction mixture was stirred at RT overnight. The reaction mixture was then diluted with ethyl acetate and the organic phase was washed twice with saturated sodium carbonate solution and once with saturated NaCl solution. After drying over magnesium sulphate, the solvent was evaporated under reduced pressure and the residue was purified using silica gel (mobile phase: dichloromethane/methanol 100:1). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 188.6 mg (74%) of the compound 2-[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione.

LC-MS (Method 1): R_(t)=1.00 min; MS (ESIpos): m/z=541 [M+H]⁺.

171.2 mg (0.32 mmol) of 2-[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]-1H-isoindole-1,3(2H)-dione were initially charged in 5.0 ml of dichloromethane, and 73.6 mg (0.73 mmol) of triethylamine were added. At 0° C., 94.9 mg (0.70 mmol) of acetoxyacetyl chloride were added, and the reaction mixture was stirred at RT overnight. The reaction mixture was diluted with ethyl acetate and the organic phase was washed twice with saturated sodium bicarbonate solution and once with sat. NaCl solution. After drying over magnesium sulphate, the solvent was evaporated under reduced pressure and the residue was purified using Biotage Isolera (silica gel, column 10 g SNAP, flow rate 12 ml/min, ethyl acetate/cyclohexane 1:3). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 159.0 mg (77%) of the compound 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate.

LC-MS (Method 1): R_(t)=1.35 min; MS (ESIpos): m/z=642 [M+H]⁺.

147.2 mg (0.23 mmol) of 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]amino)-2-oxoethyl acetate were initially charged in 4.0 ml of ethanol, and 356.2 mg (4.59 mmol) of methanamine (40% in water) were added. The reaction mixture was stirred at 50° C. overnight. The solvent was evaporated under reduced pressure and the residue was co-distilled with toluene three times. The residue was purified using silica gel (mobile phase: dichloromethane/methanol 10:1). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 67.4 mg (63%) of the title compound.

LC-MS (Method 1): R_(t)=0.91 min; MS (ESIpos): m/z=470 [M+H]⁺.

Intermediate C102 tert-butyl {(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)amino]-1-oxobutan-2-yl}carbamate

20.0 mg (32.54 μmol) (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-[(tert-butoxycarbonyl)amino]butanoic acid (intermediate C5), 10.1 mg (32.54 μmol) N-(2-aminoethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide trifluoroacetate (1:1) (intermediate L1) and 18.6 mg (48.81 μmol) HATU were dissolved in 2.5 ml DMF. 16.8 mg (23 μl, 130.15 μmol) N,N-diisopropylethylamine were added and the mixture was stirred at room temperature for 1 h. The mixture was purified directly via preparative HPLC (eluent: acetonitrile/water+0.1% TFA, gradient 35:65→95:5) followed by freeze-drying to give 15 mg (58%) of the target compound.

LC-MS (Method 1): R_(t)=1.23 min; MS (EIpos): m/z=794 [M+H]⁺.

Intermediate C103 tert-butyl [(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-({2-[({3-[(2,2-dimethyl-4,12-dioxo-3,15,18,21,24-pentaoxa-5,11-diaza-hexacosan-26-yl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}acetyl)amino]ethyl}amino)-1-oxobutan-2-yl]carbamate

15 mg (18.90 μmol) tert-butyl {(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-ethyl)amino]-1-oxobutan-2-yl}carbamate (intermediate C102) were dissolved in 500 μl acetonitrile. 12.3 mg (26.45 μmol) tert-butyl (7-oxo-21-sulfanyl-10,13,16,19-tetraoxa-6-azahenicos-1-yl)carbamate (intermediate L2) dissolved in 275 μl phosphate-buffered saline (pH=7) were added to the mixture. The pH of the solution was adjusted to pH=8 by adding some drops of 1N sodium hydroxide solution. The mixture was stirred for 30 min. at room temperature. The mixture was purified via preparative HPLC (eluent: acetonitrile/water+0.1% TFA, gradient 35:65→95:5) followed by freeze-drying to give 20 mg (84%) of the target compound.

LC-MS (Method 1): R_(t)=1.26 min; MS (EIpos): m/z=1261 [M+H]⁺.

Intermediate C104 (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl) amino]-2-{[(benzyloxy)carbonyl]amino}butanoic acid

Intermediate C52 was reductively alkylated with benzyl-(2S)-2-{[(benzyloxy)carbonyl]amino}-4-oxobutanoate in analogy to intermediate C2. Subsequently the secondary amino group was acylated with 2-chloro-2-oxoethylacetate. In the final step, the two ester groups were cleaved using 2M lithium hydroxide solution in methanol.

LC-MS (Method 1): R_(t)=1.31 min; MS (ESIpos): m/z=646 (M−H)⁻.

Intermediate C105 Benzyl N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethyl propyl}(glycoloyl)amino]butanoyl}-beta-alaninate trifluoroacetate (1:1)

200 mg of intermediate C58 were coupled with benzyl beta-alaninate in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequently the Teoc-protecting group was cleaved using 4 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 40 min to 50° C. After addition of 4 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 12): R_(t)=1.7 min; MS (ESIpos): m/z=675 (M+H)⁺.

Intermediate C106 2-(Trimethylsilyl)ethyl N-[2-({2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]-N2-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-glutaminate

151 mg (0.23 mmol) of intermediate C104 were coupled with 128 mg (0.234 mmol) of intermediate L9 in DMF in the presence of HATU and N,N-diisopropylethylamine. Subsequently the Z-protecting group was cleaved by hydrogenation over 10% palladium/activated charcoal under normal pressure.

Yield: 30% of th. over 2 steps

LC-MS (Method 1): R_(t)=1.14 min; MS (ESIpos): m/z=929 (M+H)⁺.

Intermediate C108 2-(Trimethylsilyl)ethyl N⁶—(N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-beta-alanyl)-N²-{[2-(trimethylsilyl) ethoxy]carbonyl}-L-lysinate

103 mg (0.16 mmol) of intermediate C104 were coupled with 110 mg (0.175 mmol) of 2-(Trimethyl silyl)ethyl N⁶-beta-alanyl-N²-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-lysinat (intermediate L11) in DMF in the presence of EDC, HOBT and N,N-diisopropylethylamine. Subsequently the Z-protecting group was cleaved by hydrogenation in DCM-methanol 1:1 over 10% palladium/activated charcoal under normal pressure. The title compound was obtained in a yield of 113 mg (75% over 2 steps).

LC-MS (Method 1): R_(t)=1.17 min; MS (ESIpos): m/z=957 (M+H)⁺.

Intermediate C109 dibenzyl N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethyl propyl}(glycoloyl)amino]butanoyl}-beta-alanyl-L-glutamate

The title compound was obtained by coupling of intermediate C61 with dibenzyl L-glutamate in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the Teoc protecting group using 10 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 1 h to 50° C. in trifluoroethanol.

LC-MS (Method 1): R_(t)=1.09 min; MS (ESIpos): m/z=894 (M+H)⁺.

Intermediate C110 N2-acetyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]-N6-(tert-butoxycarbonyl)-L-lysinamide

The title compound was obtained by coupling of intermediate C104 with intermediate L13 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the the Z-protecting by hydrogenation in DCM-methanol 1:1 over 10% palladium/activated charcoal under normal pressure.

LC-MS (Method 1): R_(t)=0.96 min; MS (ESIpos): m/z=826 (M+H)⁺.

Intermediate C111 2-(Trimethylsilyl)ethyl (3R,4R)-3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-fluoropyrrolidine-1-carboxylate trifluoroacetate (1:1)

To a solution of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine (108 mg, 304 μmol) (intermediate C52) in 56.0 ml DCM an mol. sieves 4 Å was added sodium triacetoxyborhydrid (90.2 mg, 426 μmol). The mixture was stirred at RT for 15 min and 2-(trimethylsilyl)ethyl (3R,4S)-3-fluoro-4-formylpyrrolidine-1-carboxylate (97.3 mg, 98% purity, 365 μmol) (Ref: WO 2014/151030A1) was then added. The reaction mixture was stirred at RT for 3.5 h and then diluted with DCM. The organic layer was washed with sat. sodium hydrogencarbonate and water.

The organic layer was then dried over sodium sulfate and evaporated. The residue was purified by preparative RP-HPLC to afford 1.39 g (24% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.15 min; MS (ESIpos): m/z=600 (M+H)⁺.

Intermediate C112 2-(Trimethylsilyl)ethyl (3R,4R)-3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]methyl}-4-fluoropyrrolidine-1-carboxylate

To a solution of 692.8 mg (0.88 mmol) 2-(trimethylsilyl)ethyl (3R,4R)-3-[({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)methyl]-4-fluoropyrrolidine-1-carboxylate trifluoroacetate (1:1) (Intermediate C111) in 8.7 ml DCM with mol. sieves 4 Å were added 295.0 mg (2.91 mmol) triethylamine and 418.9 mg (3.71 mmol) chloracetylchloride. The reaction mixture was stirred at RT for 2.5 h and then diluted with DCM. The organic layer was washed with sat. sodium hydrogencarbonate and sat. ammonium chloride. The organic layer was then dried over sodium sulfate and evaporated. The residue was diluted in 8.7 ml DCM and mol. sieves 4 Å, 295.0 mg (2.91 mmol) triethylamine and 418.9 mg (3.71 mmol) chloracetylchloride were added. The reaction mixture was stirred at RT for 3 h and was then diluted with DCM. The organic layer was washed with sat. sodium hydrogencarbonate and sat. ammonium chloride. The organic layer was then dried over sodium sulfate and evaporated to afford 691 mg (74% of theory) of the title compound which was used in the next step without further purification.

LC-MS (Method 1): R_(t)=1.78 min; MS (ESIpos): m/z=676 (M+H)⁺.

Intermediate C113 S-[2-({(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3R,4R)-4-fluoro-1-{[2-(trimethylsilyl)ethoxy]carbonyl}pyrrolidin-3-yl]methyl}amino)-2-oxoethyl]-L-cysteine

To a suspension of 203.6 mg (1.68 mmol) L-cysteine and 201.7 mg (2.40 mmol) sodium hydrogencarbonate in 0.95 mL of water was added a solution of 170.0 mg (0.24 mmol) 2-(trimethylsilyl)ethyl (3R,4R)-3-{[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]methyl}-4-fluoropyrrolidine-1-carboxylate (intermediate C112) in 9.5 mL iso-propanol and 438.5 mg (2.40 mmol) 1,8-diazabicyclo(5.4.0)undec-7-ene. The reaction mixture was stirred at 50° C. for 3 h. The reaction mixture was then diluted with ethyl acetate and the organic layer was washed with sat. sodium hydrogencarbonate and brine. The organic layer was then dried over sodium sulfate and the solvent was evaporated to afford 152 mg (83% of theory) of the title compound which was used in the next step without further purification.

LC-MS (Method 1): R_(t)=1.26 min; MS (ESIpos): m/z=762 (M+H)⁺.

Intermediate L1 Trifluoroacetic acid/N-(2-aminoethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide (1:1)

The title compound was prepared by classical methods of peptide chemistry starting from commercially available (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid and tert-butyl (2-aminoethyl)carbamate. HPLC (Method 11): R_(t)=0.19 min;

LC-MS (Method 1): R_(t)=0.17 min; MS (ESIpos): m/z=198 (M+H)⁺.

Intermediate L2 Tert-butyl (7-oxo-21-sulfanyl-10,13,16,19-tetraoxa-6-azahenicos-1-yl)carbamate

356 mg (1.757 mmol) tert-butyl (5-aminopentyl)carbamate, 496 mg (1.757 mmol) 1-sulfanyl-3,6,9,12-tetraoxapentadecan-15-oic acid and 801 mg (2.108 mmol) HATU were dissolved in 5.95 ml DMF. The reaction mixture was cooled with an ice bath and 681 mg (920 μl, 5.272 mmol) N,N-diisopropylethylamine was added. The mixture was stirred at room temperature for 4 h and stored overnight in a refrigerator at 4° C. The mixture was purified directly via preparative HPLC (eluent: acetonitrile/water+0.1% TFA, gradient 20:80→80:20) followed by freeze-drying to give 255 mg (29%) of the target compound.

LC-MS (Method 1): R_(t)=0.87 min; MS (EIpos): m/z=467 [M+H]⁺.

Intermediate L3 N²-acetyl-N⁶-[(benzyloxy)carbonyl]-L-lysine

This intermediate was obtained by reacting benzyl carbonochloridate with N²-acetyl-L-lysine.

LC-MS (Method 1): R_(t)=0.71 min; MS (ESIpos): m/z=323 (M+H)⁺.

Intermediate L4 N²-acetyl-N⁶-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-lysine

This intermediate was obtained by reacting 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy) pyrrolidine-2,5-dione with N2-acetyl-L-lysine.

LC-MS (Method 1): R_(t)=0.87 min; MS (ESIpos): m/z=333 (M+H)⁺.

Intermediate L5 9H-Fluoren-9-ylmethyl {(5S)-5-acetamido-6-[(2-aminoethyl)amino]-6-oxohexyl}carbamate trifluoroacetate (1:1)

This intermediate was obtained by reacting commercially available N²-acetyl-N⁶-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine with tert-butyl (2-aminoethyl)carbamate and subsequent removal of the Boc-group with trifluoroacetic acid.

LC-MS (Method 13): R_(t)=0.87 min; MS (ESIpos): m/z=453 (M+H)⁺.

Intermediate L6 2-(Trimethylsilyl)ethyl 3-{[(benzyloxy)carbonyl]amino}-D-alaninate trifluoroacetate (1:1)

The title compound was prepared starting from 3-{[(benzyloxy)carbonyl]amino}-N-(tert-butoxycarbonyl)-D-alanine using classical peptide chemistry methods. Esterification with 2-(trimethylsilyl)ethanol using EDC/DMAP followed by deprotection of the Boc group by TFA provided 405 mg (58% over 2 steps) of the title compound.

LC-MS (Method 1): R_(t)=0.75 min; MS (ESIpos): m/z=339 (M+H)⁺.

Intermediate L7 N²-acetyl-N⁶-[(benzyloxy)carbonyl]-L-lysyl-L-alanyl-L-alanyl-L-asparagine

The synthesis of the title compound started with coupling of N-[(benzyloxy)carbonyl]-L-alanyl-L-alanine and tert-butyl L-asparaginate in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the Z-protecting group by hydrogenation in methanol with 10% palladium on activated charcoal. The intermediate obtained was then coupled with intermediate L3 in DMF in the presence of HATU and N,N-diisopropylethylamine and in the final step the tert-butylester group was cleaved with trifluoroacetic acid in DCM.

LC-MS (Method 1): R_(t)=0.59 min; MS (ESIpos): m/z=579 (M+H)⁺.

Intermediate L8 N²-acetyl-N⁶-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-lysyl-L-alanyl-L-alanyl-L-asparagine

The synthesis of the title compound started with coupling of N-[(benzyloxy)carbonyl]-L-alanyl-L-alanine and tert-butyl L-asparaginate in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the Z-protecting group by hydrogenation in methanol over 10% palladium/activated charcoal under normal pressure. The intermediate obtained was then coupled with intermediate L4 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequently both protecting groups were removed under stirring for 1h in 7.5% trifluoroacetic acid in DCM. In the final step the amino group was again protected using 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in the presence of N,N-diisopropylethylamine in DMF.

LC-MS (Method 1): R_(t)=0.71 min; MS (ESIpos): m/z=589 (M+H)⁺.

Intermediate L9 2,2-Dimethylpropanoic acid-2-(trimethylsilyl)ethyl-N-(2-aminoethyl)-N²-{[2-(trimethylsilyl) ethoxy]carbonyl}-L-glutaminate (1:1)

First (4S)-5-tert-butoxy-4-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid was coupled with benzyl-(2-aminoethyl)carbamate in the presence of HATU and N,N-diisopropylethylamine. Subsequently both, the Boc group and the tert-butylester group were cleaved using trifluoro acetic acid. Then first the amino group was protected again by reaction with trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in DMF/water in the presence of N,N-diisopropylethylamine and then the carboxy group was esterified with 2-(trimethylsilyl)ethanol in DCM using EDC/DMAP. In the final step the Z-protecting group was removed by hydrogenation over 10% palladium/activated charcoal under normal pressure and the title compound was obtained and purified by HPLC.

LC-MS (Method 1): R_(t)=0.82 min; MS (ESIpos): m/z=434 (M+H)⁺.

Intermediate L10 tert-butyl N-[(benzyloxy)carbonyl]-L-alanyl-L-alanyl-L-asparaginate

The title compound was obtained by coupling of N-[(benzyloxy)carbonyl]-L-alanyl-L-alanine with tert-butyl L-asparaginate in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the tert-butylester group by trifluoroacetic acid in DCM.

LC-MS (Method 1): R_(t)=0.5 min; MS (ESIpos): m/z=409 (M+H)⁺.

Intermediate L11 2-(trimethylsilyl)ethyl N⁶-beta-alanyl-N²-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-lysinate

The title compound was synthesized with classical methods of peptide chemistry starting with the coupling of N-(tert-butoxycarbonyl)-beta-alanine with 2-(trimethylsilyl)ethyl N²-[(benzyloxy)carbonyl]-L-lysinate with HATU and N,N-diisopropylethylamine, removal of the Z-protecting group by hydrogenation over 10% palladium/activated charcoal under normal pressure, introduction of the trimethylsilyl-ethyloxy carbonyl(Teoc)-protecting group with 1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione and finally mild removal of the Boc-protecting group by stirring for 45 minutes in a 7.5% solution of trifluoro acetic acid in dichloromethane.

LC-MS (Method 1): R_(t)=0.83 min; MS (ESIpos): m/z=462 (M+H)⁺.

Intermediate L12 Benzyl (2S)-5-({(5S)-5-acetamido-6-[(2-aminoethyl)amino]-6-oxohexyl}amino)-2-{[(benzyloxy)carbonyl]amino}-5-oxopentanoate trifluoroacetate (1:1)

The title compound was synthesized with classical methods of peptide chemistry starting with coupling of tert-butyl (2-aminoethyl)carbamate with intermediate L3 with HATU and N,N-diisopropylethylamine, removal of the Z-protecting group by hydrogenation in DCM/methanol 1:1 over 10% palladium/activated charcoal under normal pressure, coupling of the obtained intermediate with (4S)-5-(benzyloxy)-4-{[(benzyloxy)carbonyl]amino}-5-oxopentanoic acid with HATU and N,N-diisopropylethylamine and finally removal of the Boc-protecting group by stirring for 1 h in a 25% solution of trifluoro acetic acid in dichloromethane.

LC-MS (Method 1): R_(t)=0.66 min; MS (ESIpos): m/z=584 (M+H)⁺.

Intermediate L13 Benzyl (2S)-5-({(5S)-5-acetamido-6-[(2-aminoethyl)amino]-6-oxohexyl}amino)-2-{[(benzyloxy)carbonyl]amino}-5-oxopentanoate trifluoroacetate (1:1)

The title compound was synthesized with classical methods of peptide chemistry starting with coupling of N2-acetyl-N6-(tert-butoxycarbonyl)-L-lysine with benzyl (2-aminoethyl)carbamate hydrochloride (1:1) with HATU and N,N-diisopropylethylamine and subsequent removal of the Z-protecting group by hydrogenation in DCM/methanol 1:1 over 10% palladium/activated charcoal under normal pressure. LC-MS (Method 1): R_(t)=0.43 min; MS (ESIpos): m/z=331 (M+H)⁺.

Intermediate L14 N-(pyridin-4-ylacetyl)-L-alanyl-L-alanyl-L-asparagine trifluoroacetate (1:1)

The title compound was synthesized applying classical methods of peptide chemistry starting with coupling of pyridin-4-ylacetic acid hydrochloride (1:1) with commercially available tert-butyl L-alanyl-L-alaninate hydrochloride (1:1) with HATU and N,N-diisopropylethylamine and subsequent removal of the tert-butylester with trifluoroacetic acid in DCM. The obtained intermediate was coupled with tert-butyl L-asparaginate in the presence of HATU and N,N-diisopropylethylamine and finally the tert-butylester with cleaved with trifluoroacetic acid in DCM.

LC-MS (Method 1): R_(t)=0.15 min; MS (ESIpos): m/z=394 (M+H)⁺.

Intermediate L15 2-(Trimethylsilyl)ethyl (3-oxopropyl)carbamate

434.4 mg (5.78 mmol) of 3-amino-1-propanol and 1.50 g (5.78 mmol) of 2-(trimethylsilyl)ethyl 2,5-dioxopyrrolidine-1-carboxylate were dissolved in 10.0 ml of dichloromethane, 585.3 mg (5.78 mmol) of triethylamine were added and the mixture was stirred at RT overnight. The reaction mixture was diluted with dichloromethane and the organic phase was washed with water and saturated sodium bicarbonate solution and then dried over magnesium sulphate. The solvent was evaporated under reduced pressure. The residue 2-(trimethylsilyl)ethyl (3-hydroxypropyl)carbamate (996.4 mg, 79% of theory) was dried under high vacuum and used without further purification in the next step of the synthesis.

807.0 mg (3.68 mmol) of 2-(trimethylsilyl)ethyl (3-hydroxypropyl)carbamate were initially charged in 15.0 ml of chloroform and 15.0 ml of 0.05 N potassium carbonate/0.05 N sodium bicarbonate solution (1:1). 102.2 mg (0.37 mmol) of tetra-n-butylammonium chloride, 736.9 mg (5.52 mmol) of N-chlorosuccinimide and 57.5 mg (0.37 mmol) of TEMPO were then added and the reaction mixture was stirred vigorously at RT overnight. The reaction mixture was diluted with dichloromethane and the organic phase was washed with water and saturated NaCl solution. The organic phase was dried over magnesium sulphate and the solvent was evaporated under reduced pressure. The residue was dried under high vacuum and used without further purification in the next step of the synthesis (890.3 mg).

Intermediate L16 2,2-dimethyl-4,11-dioxo-3,15,18,21,24-pentaoxa-5,12-diazaheptacosan-27-oic acid

The title compound was obtained by coupling of methyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate with 6-[(tert-butoxycarbonyl)amino]hexanoic acid in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent saponification of the ester group using 5 equivalents of lithium hydroxide in THF: Water (1:1) at room temperature for 1 h.

LC-MS (Method 12): Rt=1.25 min; MS (ESIpos): m/z=479 [M+H]+

Intermediate L17 2-(trimethylsilyl)ethyl 3-{[N²-acetyl-N⁶-(tert-butoxycarbonyl)-L-lysyl]amino}-D-alaninate

The title compound was obtained by coupling of 3-amino-N-[(benzyloxy)carbonyl]-D-alanine and 2,5-dioxopyrrolidin-1-yl N²-acetyl-N⁶-(tert-butoxycarbonyl)-L-lysinate in DMF in the presence of N,N-diisopropylethylamine, followed by coupling of the carboxylic acid group with 2-(trimethylsilyl)ethanol in acetonitrile in the presence of pyridine and 1,3-dicyclohexylcarbodiimide and subsequent deprotection of the benzyloxycarbonyl group by hydrogenation in methanol in presence of 10% Pd/C at room temperature for 2 h.

LC-MS (Method 1): R_(t)=0.70 min; MS (ESIpos): m/z=475 (M+H)⁺.

Intermediate L81 benzyl {2-[(2-aminoethyl)sulfonyl]ethyl}carbamate trifluoroacetate (1:1)

250 mg (1.11 mmol) 2,2′-Sulfonyldiethanamine were coupled with 92.3 mg (0.37 mmol) 1-{[(Benzyloxy)carbonyl]oxy}pyrrolidin-2,5-dion in the presence of N, N-diisopropylethylamine in DMF. After HPLC purification 70 mg (47% d. Th.) of the title compound were obtained.

LC-MS (Method 12): R_(t)=0.64 min; MS (ESIpos): m/z=257.11 (M+H)⁺.

Intermediate L108 N²-Acetyl-N-(2-aminoethyl)-N⁶-(tert-butoxycarbonyl)-L-lysinamide

The title compound was obtained by coupling of N²-acetyl-N⁶-(tert-butoxycarbonyl)-L-lysine and benzyl (2-aminoethyl)carbamate hydrochloride (1:1) in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent deprotection of the benzyloxycarbonyl group by hydrogenation in dichloromethane/methanol 1:1 in presence of 10% Pd/C at room temperature for 1 h.

LC-MS (Method 1): R_(t)=0.43 min; MS (ESIpos): m/z=331 (M+H)⁺.

Intermediate F1 1-{[1-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2- yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]amino}-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl]sulfanyl}-N-(5-aminopentyl)-3,6,9,12-tetraoxapentadecan-15-amide dihydrochloride

19 mg (15.07 μmol) tert-butyl [(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-({2-[({3-[(2,2-dimethyl-4,12-dioxo-3,15,18,21,24-pentaoxa-5,11-diazahexacosan-26-yl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}acetyl)amino]ethyl}amino)-1-oxobutan-2-yl]carbamate (intermediate C103) were dissolved in 300 μl DCM. 113 μl hydrogen chloride solution 4M in dioxane were added and the mixture was stirred at room temperature for 1h. Additional 100 hydrogen chloride solution 4 M in dioxane were added and the mixture was again stirred for 1h.

The solvent was evaporated and the residue was solidified by freeze-drying from a mixture of acetonitrile and water to yield 11 mg (64%) of the target compound.

LC-MS (Method 4): R_(t)=5.86 min; MS (EIpos): m/z=1060 [M+H]⁺.

Intermediate F2 N²-acetyl-L-lysyl-L-valyl-N-{(1S)-3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-carboxypropyl}-L-alaninamide trifluoroacetate (1:1)

The title compound was prepared using classical methods well known in peptide synthesis starting from methyl-(2S)-4-[(acetoxyacetyl){(1R)-1-[1-benzyl-4-(2,5-difluorphenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoat (intermediate C75). First the Teoc group was cleaved using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 2 h to 50° C. in trifluoroethanol. Subsequently the deprotected intermediate was reacted with N-[(benzyloxy)carbonyl]-L-valyl-L-alanine in the presence of HATU and N,N-diisopropylethylamine in DMF. In the next step the Z-protecting group was removed by hydrogenation over 10% palladium/activated charcoal under normal pressure. The intermediate obtained was reacted with intermediate L3 in the presence of HATU and N,N-diisopropylethylamine in DMF. Susequent ester cleavage with 2M Lithiumhydroxide solution in water/THF 2:1 and final removal of the Z-protecting group by hydrogenation over 10% palladium/activated charcoal under normal pressure gave the title compound.

LC-MS (Method 1): R_(t)=0.9 min; MS (ESIpos): m/z=854 (M+H)⁺.

Intermediate F3 N²-acetyl-L-lysyl-L-valyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]propyl}-L-alaninamide trifluoroacetate (1:1)

The title compound was synthesized using classical methods well known in peptide synthesis starting from N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethyl propyl}-2-hydroxyacetamide (intermediate C101) by coupling with benzyloxy)carbonyl]-L-valyl-L-alanine in the presence of HATU and N,N-diisopropylethylamine in DMF. In the next step the Z-protecting group was removed by hydrogenation over 10% palladium/activated charcoal under normal pressure. The intermediate obtained was reacted with intermediate L3 in the presence of HATU and N,N-diisopropylethylamine in DMF. Finally by hydrogenation over 10% palladium/activated charcoal under normal pressure the title compound was obtained.

LC-MS (Method 1): R_(t)=0.90 min; MS (ESIpos): m/z=810 (M+H)⁺.

Intermediate F4 3-[(N²-acetyl-L-lysyl)amino]-N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-D-alanine trifluoroacetate (1:1)

25 mg (0.026 mmol) of intermediate C74 were dissolved in 3.75 mL DMF and coupled with 13 mg (0.031 mmol) of commercially available N2-acetyl-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine in the presence of 12 mg (0.031 mmol) HATU and 3 equivalents of N, N diisopropylethylamine. In the second step the Fmoc protecting group was cleaved with 100 equivalents of DABCO in 5 ml DMF. Finally the Teoc protecting group was cleaved with 6 equivalents of zinc chloride under heating for 2 h to 50° C. in trifluoroethanol. After HPLC purification 4.5 mg (20%) of the title compound were obtained.

LC-MS (Method 1): R_(t)=0.76 min; MS (ESIpos): m/z=770 (M+H)⁺.

Intermediate F5 N²-acetyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]-L-lysinamide trifluoroacetate (1:1)

The title compound was obtained by coupling of (2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic acid (Intermediate C53) with Intermediate L5 and subsequent removal of the Fmoc protecting groups with 100 equivalents of DABCO in DMF.

LC-MS (Method 12): R_(t)=1.11 min; MS (EIpos): m/z=724.40 [M+H]⁺.

Intermediate F6 3-({15-[(N²-acetyl-L-lysyl)amino]-4,7,10,13-tetraoxapentadecan-1-oyl}amino)-N-{(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}-D-alanine trifluoroacetate (1:1)

In the initial step intermediate C74 was coupled with commercially available 9H-fluoren-9-ylmethyl {15-[(2,5-dioxopyrrolidin-1-yl)oxy]-15-oxo-3,6,9,12-tetraoxapentadec-1-yl}carbamate in the presence of N, N diisopropylethylamine in DMF. Subsequently the Fmoc protecting group was cleaved with 100 equivalents of DABCO in DMF and the intermediate obtained was coupled with L4 in the presence of 1.1 equivalents of HATU and 3 equivalents of N, N diisopropylethylamine in DMF. Finally the Teoc protecting groups and the trimethylsilylethylester were cleaved with 12 equivalents of zinc chloride under heating for 1 h to 50° C. in trifluoroethanol. After HPLC purification 2.3 mg of the title compound were obtained.

LC-MS (Method 1): R_(t)=0.72 min; MS (EIpos): m/z=1017 [M+H]⁺.

Intermediate F7 N²-acetyl-L-lysyl-L-alanyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(2-carboxyethyl)amino]-1-oxobutan-2-yl}-L-aspartamide

The title compound was obtained by coupling of intermediate C105 with intermediate L7 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent simultaneous removal of the Z-protecting group and the benzyl ester by hydrogenation in DCM-methanol 1:1 with 10% palladium on activated charcoal and HPLC purification.

LC-MS (Method 1): R_(t)=0.82 min; MS (ESIpos): m/z=1011 (M+H)⁺.

Intermediate F8 N²-acetyl-L-lysyl-L-alanyl-L-alanyl-N¹-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-{[2-(L-gamma-glutamylamino)ethyl]amino}-1-oxobutan-2-yl]-L-aspartamide trifluoroacetate (1:1)

The title compound was obtained by coupling of intermediate C106 with intermediate L8 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent simultaneous removal of all protecting groups using 10 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 6 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 1): R_(t)=0.72 min; MS (ESIpos): m/z=1111 (M+H)⁺.

Intermediate F9 N²-acetyl-L-lysyl-L-alanyl-L-alanyl-N¹-{(2S)-1-[(3-{[(5S)-5-amino-5-carboxypentyl]amino}-3-oxopropyl)amino]-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-oxobutan-2-yl}-L-aspartamide trifluoroacetate (1:1)

The title compound was obtained by coupling of intermediate C108 with intermediate L10 in DMF in the presence of HATU and N,N-diisopropylethylamine, subsequent removal of the Z-protecting group by hydrogenation in DCM/methanol 1:1 over 10% palladium/activated charcoal under normal pressure, then coupling with intermediate L4 in DMF in the presence of HATU and N,N-diisopropylethylamine and finally simultaneous removal of all protecting groups using 12 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 2 h to 50° C. After addition of 12 equivalents of EDTA the product is purified by HPLC.

LC-MS (Method 12): R_(t)=11.7 min; MS (ESIneg): m/z=1137 (M−H)⁻.

Intermediate F10 N²-acetyl-L-lysyl-L-alanyl-L-alanyl-N¹-{(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-[(3-{[(1S)-1,3-dicarboxypropyl]amino}-3-oxopropyl)amino]-1-oxobutan-2-yl}-L-aspartamide trifluoroacetate (1:1)

The title compound was obtained by coupling of intermediate C109 with intermediate L8 in DMF in the presence of HATU and N,N-diisopropylethylamine, subsequent removal of the benzyl ester protecting groups by hydrogenation in methanol over 10% palladium/activated charcoal under normal pressure and finally removal of the Teoc protecting group using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 6 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 12): R_(t)=1.31 min; MS (ESIpos): m/z=1140 (M+H)⁺.

Intermediate F11 N²-acetyl-L-lysyl-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cysteine trifluoroacetate (1:2)

To a solution of S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-L-cysteine trifluoroacetate (1:1) (20.0 mg, 24.1 μmol) (intermediate C71) and N²-acetyl-N⁶-(tert-butoxycarbonyl)-L-lysine (9.02 mg, 31.3 μmol) in acetonitrile (2.0 ml) were added N,N-diisopropylethylamine (34 μl, 190 μmol) and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (19 μl, 50% purity, 31 μmol). The mixture was stirred over night at room temperature and purified by preparative RP-HPLC (column: Reprosil 250×30; 10μ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 5.2 mg (22% of theory) of N²-acetyl-N⁶-(tert-butoxycarbonyl)-L-lysyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-L-cysteine. The product contained around 15% of its epimer.

To a solution of N²-acetyl-N⁶-(tert-butoxycarbonyl)-L-lysyl-S-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-L-cysteine (61.6 mg, 62.4 μmol) in 2,2,2-Trifluorethanol (6.0 ml) was added Zinkchlorid (51.0 mg, 374 μmol) and the mixture was stirred at 50° C. for 1 h. Zinkchlorid (51.0 mg, 374 μmol) was then added and the mixture was stirred at 50° C. for 1 h. The mixture was stirred for 5 min with EDTA (218 mg, 748 μmol) and purified by preparative RP-HPLC (column: Reprosil 250×30; 10μ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvents were evaporated under reduced pressure and the residue was lyophilized. This gave 41.6 mg (69% of theory) of the title compound.

LC-MS (Method 12): R_(t)=1.32 min; MS (ESIneg): m/z=741 [M−H]⁻

Intermediate F12 N-(6-aminohexanoyl)-S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3S,4R)-4-fluoropyrrolidin-3-yl]methyl}amino)-2-oxoethyl]-L-cysteine trifluoroacetate (1:1)

To a solution of 6-[(tert-butoxycarbonyl)amino]hexanoic acid (29.2 mg, 126 μmol) in 1.4 ml DMF were added N,N-diisopropylethylamine (21 μl, 120 μmol) and HATU (46.2 mg, 122 μmol). The reaction mixture was stirred at RT for 10 min and a solution of S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3R,4R)-4-fluoro-1-{[2-(trimethylsilyl)ethoxy]carbonyl}pyrrolidin-3-yl]methyl}amino)-2-oxoethyl]-L-cysteine (37.0 mg, 48.6 μmol) (intermediate C107) in 1.4 ml DMF was then added. The reaction mixture was then stirred overnight at RT. Water and DCM were then added and the organic phase was separated, further washed with water, dried over magnesium sulfate and evaporated to afford 49.7 mg (85% of theory) of S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3R,4R)-4-fluoro-1-{[2-(trimethylsilyl)ethoxy]carbonyl}pyrrolidin-3-yl]methyl}amino)-2-oxoethyl]-N-{6-[(tert-butoxycarbonyl)amino]hexanoyl}-L-cysteine which was used in the next step without further purification.

LC-MS (Method 1): Rt=1.47 min; MS (ESIpos): m/z=974 [M+H]⁺

To a solution of S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{[(3R,4R)-4-fluoro-1-{[2-(trimethylsilyl)ethoxy]carbonyl}pyrrolidin-3-yl]methyl}amino)-2-oxoethyl]-N-{6-[(tert-butoxycarbonyl)amino]hexanoyl}-L-cysteine (49.7 mg, 81% purity, 41.4 μmol) in 3.5 ml trifluorethanol, was added (45.2 mg, 332 μmol) and the reaction mixture was stirred at 50° C. for 2 h. EDTA (96.9 mg, 332 μmol) was then added and the resulting mixture was stirred at RT for 15 min. Ethyl acetate was then added and the organic phase was washed with water and brine, dried over magnesium sulfate and evaporated. The residue was purified by preparative RP-HPLC (MeCN/water, 0.1% TFA) to afford 4.5 mg (13% of theory) of the title compound.

LC-MS (Method 1): Rt=0.79 min; MS (ESIpos): m/z=730 [M+H]⁺

Intermediate F13 N-(pyridin-4-ylacetyl)-L-alanyl-L-alanyl-N¹-{(2S)-1-({2-[(N²-acetyl-L-lysyl)amino]ethyl}amino)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-1-oxobutan-2-yl}-L-aspartamide trifluoroacetate (1:1)

The title compound was obtained by coupling of intermediate C110 with intermediate L14 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the Boc group using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 0.5 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 1): R_(t)=0.76 min; MS (ESIpos): m/z=1101 (M+H)⁺.

Intermediate F14 N²-acetyl-N-(2-{[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]sulfonyl}ethyl)-L-lysinamide trifluoroacetate (1:2)

Initially intermediate L81 was coupled with intermediate C58 in the presence of HATU and N,N-diisopropylethylamine. Subsequently the Z protecting group was removed by hydrogenation in DCM/methanol 1:1 over 10% palladium/activated charcoal under normal pressure. The intermediate obtained was coupled with intermediate L4 in the presence of HATU and N,N-diisopropylethylamine. Finally the Teoc protecting groups were cleaved using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 2 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 12): R_(t)=0.7 min; MS (ESIpos): m/z=818 (M+H)⁺.

Intermediate F15 N²-acetyl-N-{2[(3-{[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[pyrrolidin-3-ylmethyl]amino)-2-oxoethyl]sulfanyl}propanoyl)amino]ethyl}-L-lysinamide trifluoroacetate (1:2) (Isomer 1)

The title compound was obtained by coupling of intermediate C95 with intermediate L108 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the Boc groups using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 1 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 1): R_(t)=0.74 min; MS (ESIpos): m/z=796 (M+H)⁺.

Intermediate F16 N-(15-amino-4,7,10,13-tetraoxapentadecan-1-oyl)-S-[2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}[pyrrolidin-3-ylmethyl]amino)-2-oxoethyl]cystein trifluoroacetat (1:2) (Isomer 1)

The title compound was obtained by coupling of intermediate C90 with 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-oic acid in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the Boc groups using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 3 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 1): R_(t)=0.80 min; MS (ESIpos): m/z=846 (M+H)⁺.

Intermediate F17 N-(22-amino-17-oxo-4,7,10,13-tetraoxa-16-azadocosan-1-oyl)-S-{2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-L-cystein trifluoroacetat (1:2)

The title compound was obtained by coupling of intermediate C71 with intermediate L16 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the protecting groups using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 1 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 1): R_(t)=0.78 min; MS (ESIpos): m/z=933 (M+H)⁺.

Intermediate F18 3-[(N²-acetyl-L-lysyl)amino]-N-[3-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)propanoyl]-D-alanin trifluoroacetat (1:2)

The title compound was obtained by coupling of intermediate C69 with intermediate L17 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the protecting groups using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 1 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 1): R_(t)=0.76 min; MS (ESIpos): m/z=814 (M+H)⁺.

Intermediate F19 N²-acetyl-N-(2-{[3-({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulfanyl)propanoyl]amino}ethyl)-L-lysinamid trifluoroacetat (1:2)

The title compound was obtained by coupling of intermediate C69 with intermediate L108 in DMF in the presence of HATU and N,N-diisopropylethylamine and subsequent removal of the protecting groups using 6 equivalents of zinc chloride in 2,2,2 trifluorethanol under heating for 1 h to 50° C. After addition of 6 equivalents of EDTA the product was purified by HPLC.

LC-MS (Method 1): R_(t)=0.79 min; MS (ESIpos): m/z=770 (M+H)⁺.

B: Preparation of Antibody/Active Compound Conjugates (ADC)

B-1. General Process for Generating Anti-TWEAKR Antibodies

The anti-TWEAKR antibodies were generated, for example, by screening of a phage display library for recombinant human TWEAKR SEQ ID NO: 138 and murine TWEAKR SEQ ID NO: 137. Particularly the antibody TPP-2090 is an important example. The antibodies obtained in this manner were reformatted into the human IgG1 format. The aglycosylated variant TPP-2090-HC-N297A was generated by introducing the mutation N297A in the heavy chain of TPP-2090 (Kabat numbering system of immunoglobulins). The aglycosylated variant TPP-2090-HC-N297Q was generated by introducing the mutation N297Q in the heavy chain of TPP-2090 (Kabat numbering system of immunoglobulins). These two antibodies were used for the working examples described here (see also WO 2015/189143 A1 and WO 2014/198817 A1). In addition, antibodies which bind to TWEAKR are known to the person skilled in the art, see, for example, WO2009/020933(A2) or WO2009140177 (A2).

SEQ ID NO: 138 (polypeptide): EQAPGTAPCSRGSSWSADLDKCMDCASCRARPHSDFCLGCAAAPPAPF RLLWPRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 137 (polypeptide): EQAPGTSPCSSGSSWSADLDKCMDCASCPARPHSDFCLGCAAAPPAHF RLLWPRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK B-2. General Process for Expressing Anti-TWEAKR Antibodies in Mammalian Cells

The antibodies, for example TPP-2090 and the aglycosylated variants TPP-2090-HC-N297A, TPP-2090-HC-N297Q, Trastuzumab-HC-N297A (equal to TPP-7510), Trastuzumab-HC-N297Q (equal to TPP-7511) were produced in transient mammalian cell cultures as described by Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007 (see AK-Example 1).

B-3. General Process for Purifying Antibodies from Cell Supernatants

The antibodies, for example TPP-2090 and the aglycosylated variants TPP-2090-HC-N297A, TPP-2090-HC-N297Q, Trastuzumab-HC-N297A (equal to TPP-7510), Trastuzumab-HC-N297Q (equal to TPP-7511) were obtained from the cell culture supernatants. The cell supernatants were clarified by centrifugation of cells. The cell supernatant was then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. To this end, the column was equilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the column was washed with about 10 column volumes of DPBS pH 7.4+500 mM sodium chloride. The antibodies were eluted in 50 mM sodium acetate pH 3.5+500 mM sodium chloride and then purified further by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

The commercially available antibody cetuximab (trade name Erbitux) was purified from the commercial product by standard chromatographic methods (protein A, preparative SEC).

The commercially available antibody trastuzumab (trade name Herceptin) was purified from the commercial product by standard chromatographic methods (protein A, preparative SEC).

Trastuzumab-HC-N297A (equal to TPP-7510) comprises a heavy chain represented by SEQ ID NO: 244. The light chain is identical with the one of Trastuzumab.

Trastuzumab-HC-N297Q (equal to TPP-7511) comprises a heavy chain represented by SEQ ID NO: 245. The light chain is identical with the one of Trastuzumab.

From the commercial product (trade name CIMAher), the antibody nimotuzumab was purified from the commercial product by standard chromatographic methods (protein A, preparative SEC).

From the commercial product (trade name Vectibix), the antibody panitumumab was purified from the commercial product by standard chromatographic methods (protein A, preparative SEC).

B-4. General Processes for Coupling to Glutamine

General Procedure A for Transglutaminase Coupling Employed in the Following Examples to Achieve a Maximum DAR of 2:

For the reactions of the ADC working examples the following antibodies were used (the following nomenclature Antibody-HC-N297Z means the antibody with the replacement of N297 (kabat numbering) by the amino acid Z in both heavy chains of the antibody, the nomenclature TPP-XXXX-HC-Q295N-HC-N297Q means the antibody with the TPP-XXXX with a replacement of the amino acid Q295 (kabat numbering) by the amino acid N in both heavy chains of the antibody and a replacement of the amino acid N297 (kabat numbering) by the amino acid Q (Kabat numbering) in both heavy chins of the antibody. The name of the antibody can be declared as name (e.g. Trastuzumab) or as TPP-XXXX (the antibody with the TPP-number XXXX):

AK_(3a): TPP-2090-HC-N297A (equal to anti-TWEAKR antibody TPP-2658)

AK_(3c): TPP-2090-HC-Q295N-HC-N297Q (equal to anti-TWEAKR antibody TPP-8825)

AK_(3d): Trastuzumab-HC-N297A (equal to TPP-7510)

To a solution of 5 mg of the aglyco variant (HC-N297A or HC-Q295N-HC-N297Q) of respective antibody in DPBS pH 7.4 (c˜5-15 mg/mL) 20 μL (6 equivalents) of a solution of the respective precursor intermediate F (10 mM in DMSO) were added. After 5 min incubation at 37° C. 50 μL (1.25 U) of a solution of recombinant microbial (bacterial) transglutaminase in water (Product number T001 from Zedira GmbH, Darmstadt, Germany) (25 U/mL) were added. The reaction mixture was incubated at 37° C. for 24 hours and subsequently diluted with DPBS pH 7.4 to a volume of 2.5 mL. The ADC solution was purified by gel filtration over PD 10-columns (Sephadex® G-25, GE Healthcare) equilibrated with DPBS-Puffer pH 7.4 which also was used for elution. Subsequently, the ADC solution was concentrated using Amicon Ultracel-30K centrifugation devices (Millipore) and re-diluted to a volume of about 2.5 mL. Finally 0.00625 μmol of b-transglutaminase blocker Zedira C100 in 12.5 μL DPBS were added. For the ADC solutions obtained the protein concentration was determined as given in the individual examples. Drug load was determined with the methods described in chapter B5. The ADC batches were characterized as pointed out in the examples.

General Procedure B for Transglutaminase Coupling Employed in the Following Examples to Achieve a Maximum DAR of 4:

For the reactions of the ADC working examples the following antibodies were used (the following nomenclature as used above):

AK_(3b): TPP-2090-HC-N297Q (equal to anti-TWEAKR antibody TPP-5442)

AK_(3e): Trastuzumab-HC-N297Q (equal to TPP-7511)

To a solution of 5 mg of the aglyco variant (HC-N297Q) of respective antibody in DPBS pH 7.4 (c˜5-15 mg/mL) 16-24 equivalents of a solution of the respective precursor intermediate F (10 mM in DMSO) were added. After 5 min incubation at 37° C. 400 μL (10 U) of a solution of recombinant microbial (bacterial) transglutaminase in water (Product number T001 from Zedira GmbH, Darmstadt, Germany) (25 U/mL) were added. The reaction mixture was incubated at 37° C. for 24 hours and subsequently diluted with DPBS pH 7.4 to a volume of 2.5 mL. The ADC solution was purified by gel filtration over PD 10-columns (Sephadex® G-25, GE Healthcare) equilibrated with DPBS-Puffer pH 7.4 which also was used for elution. Subsequently, the ADC solution was concentrated using Amicon Ultracel-30K centrifugation devices (Millipore) and re-diluted to a volume of about 2.5 mL. Finally 0.1 μmol of b-transglutaminase blocker Zedira C100 in 200 μL DPBS were added. For the ADC solutions obtained the protein concentration was determined as given in the individual examples. Drug load was determined with the methods described in chapter B7. The ADC batches were characterized as pointed out in the examples.

General Procedure C for Transglutaminase Coupling in Larger Scale Employed in the Following Examples to Achieve a Maximum DAR of 2:

To a solution of 30 mg of the aglyco variant (HC-N297A or HC-Q295N-HC-N297Q) of respective antibody in DPBS pH 7.4 (c˜5-15 mg/mL) 6 equivalents of a solution of the respective precursor intermediate F (10 mM in DMSO) were added. After 5 min incubation at 37° C. 200 μL (7.5 U) of a solution of recombinant microbial (bacterial) transglutaminase in water (Product number T001 from Zedira GmbH, Darmstadt, Germany) (25 U/mL) were added. The reaction mixture was incubated at 37° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and the transglutaminase from the ADC, and finally concentrated using Amicon Ultracel-30K centrifugation devices (Millipore) to final concentrations between 5-25 mg/mL. The solution was then sterile filtered.

The respective protein concentrations, stated in the working examples, of the ADC solutions were determined. Drug load was determined with the methods described in chapter B5. The ADC batches were characterized as pointed out in the examples.

General Procedure D for Transglutaminase Coupling in Larger Scale Employed in the Following Examples to Achieve a Maximum DAR of 4:

To a solution of 30 mg of the aglyco variant (HC-N297Q) of respective antibody in DPBS pH 7.4 (c˜5-15 mg/mL) 16-24 equivalents of a solution of the respective precursor intermediate F (10 mM in DMSO) were added. After 5 min incubation at 37° C. 2400 μL (60 U) of a solution of recombinant microbial (bacterial) transglutaminase in water (Product number T001 from Zedira GmbH, Darmstadt, Germany) (25 U/mL) were added. The reaction mixture was incubated at 37° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and the transglutaminase from the ADC, and finally concentrated using Amicon Ultracel-30K centrifugation devices (Millipore) to final concentrations between 5-25 mg/mL. The solution was then sterile filtered.

The respective protein concentrations, stated in the working examples, of the ADC solutions were determined. Drug load was determined with the methods described in chapter B5. The ADC batches were characterized as pointed out in the examples.

B-5. Determination of the Antibody, the Toxophor Loading and the Determination of Conjugation Site

For protein identification in addition to molecular weight determination after deglycosylation and/or denaturing, a tryptic digestion was carried out, which, after denaturing, reduction and derivatization, confirms the identity of the protein via the tryptic peptides found. Furthermore this approach could be used for the identification of the coupling site by detection of conjugated tryptic peptides.

The toxophor loading of the PBS buffer solutions obtained of the conjugates described in the working example was determined as follows:

Determination of toxophor loading of glutamine-linked ADCs was carried out by mass spectrometric determination of the molecular weights of the individual conjugate species. Here, the sample was acidified and after HPLC separation/desalting over a short C4 column (GromSil 300 Butyl-1 ST, 5 μm, 5 mm×500 μm), analysed by mass spectrometry using an ESI-MicroTofQ System (Bruker Daltonik). All spectra over the signal in the TIC (Total Ion Chromatogram) were added and the molecular weight of the different conjugate species was calculated based on MaxEnt deconvolution. The DAR (=drug/antibody ratio) was then calculated out of the sum of toxophor number weighted coupled species divided by the sum of the singly weighted integration results of each species.

In addition, based on the species distribution the homogeneity (prercentage of D2>85% for DAR2) of the bTG coupling method could be exemplary demonstrated comparing four different preparations of example 5A in the following table.

TABLE distribution comparison of four different preparations of example 5A (DAR2) Example 5A Conc. % distribution Prep. No [mg/mL] DAR D0 D1 D2 prep. 4  1.6 1.9 0.5 9.8 89.7 prep. 6  1.4 2.0 0.3 2.9 96.8 prep. 7  1.6 1.9 0.5 12.0 87.5 prep. 11 1.5 1.9 0.5 7.9 91.6

A comparable homogeneous distribution can be received with other bTG coupled Toxophor-Linker constructs. This was exemplary demonstrated at four different ADC compounds in the following table.

TABLE distribution of four different ADC examples (DAR2) Conc. % distribution Example [mg/mL] DAR D0 D1 D2 D3  5A 1.4 2.0 0.3 2.9 96.8 13A 2.1 1.8 2.4 12.8 84.8  8A 1.7 1.9 8.0 90.8 1.2  9A 1.8 2.0 7.7 89.7 2.6

Based on a N297Q substitution within an Antibody a bTG based coupling with a result of DAR4 is possible. The homogeneity of this ADC's (percentage of D4>70%) and transferability on different antibodies was exemplary demonstrated at three different ADC compounds in the following table.

TABLE distribution of two different ADC examples (DAR4) Conc. % distribution Example [mg/mL] DAR D0 D1 D2 D3 D4 D5 D6  6a4 1.63 4.1 0.8 3.4 77.6 18.2 10e4 1.56 3.9 2.4 6.7 89.1 1.8

Alternatively, the toxophor loading of glutamine-linked conjugates was determined by reversed-phase chromatography of the reduced and denatured ADCs. Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADC solution (1 mg/ml, 50 μl). The mixture was incubated at 55° C. for one hour and analysed by HPLC.

HPLC analysis was carried out on an Agilent 1260 HPLC system with detection at 220 nm. A Polymer Laboratories PLRP-S polymeric reversed-phase column (catalogue number PL1912-3802) (2.1×150 mm, 8 μm particle size, 1000 Å) was used at a flow rate of 1.2 ml/min with the following gradient: 0 min, 31% B; 1 min, 31% B; 14 min, 38% B; 16 min, 95% B. Mobile phase A consisted of 0.05% trifluoroacetic acid (TFA) in water, mobile phase B of 0.05% trifluoroacetic acid in acetonitrile.

The detected peaks were assigned by retention time comparison with the light chain (L0) and the heavy chain (H0) of the non-conjugated antibody. Peaks detected exclusively in the conjugated sample were assigned to the heavy chains with one or two toxophors (H1, H2).

Average loading of the antibody with toxophors was calculated from the peak areas determined by integration as double the sum of the HC-Load and the LC-load, whereas the HC-load is the sum of toxophor number weighted integration results of all heavy chain (HC)-peaks divided by the sum of the singly weighted integration results of the HC-peaks and whereas the LC-load is the sum of toxophor number weighted integration results of the light chain (LC)-peaks divided by the sum of the singly weighted integration results of all LC peaks.

B-6. Checking the Antigen-Binding of the ADCs

The capability of the binder of binding to the target molecule was checked after coupling had taken place. The person skilled in the art is familiar with multifarious methods which can be used for this purpose; for example, the affinity of the conjugate can be checked using ELISA technology or surface plasmon resonance analysis (BIAcore™ measurement). The conjugate concentration can be measured by the person skilled in the art using customary methods, for example for antibody conjugates by protein determination. (see also Doronina et al.; Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007; 1102:616-623).

WORKING EXAMPLES ADCS

The following examples were synthesized following general procedure C for transglutaminase coupling (see chapter B-4):

Example 1A

Here, 2250 μL DPBS pH 7.4, 200 μL of a solution of intermediate F1 (10 mM in DMSO), and 50 μL of a solution of recombinant microbial (bacterial) transglutaminase (Product number T001 from Zedira GmbH, Darmstadt, Germany) (100 U/mL) were added to 2500 μL of a solution of the antibody TPP-2090-HC-N297A (2 mg/mL). The reaction mixture was incubated at 37° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and the transglutaminase from the ADC, and finally concentrated using Amicon Ultracel-30K centrifugation devices (Millipore).

Protein concentration: 1.65 mg/ml

Drug/mAb ratio: 1.9

Example 1A4

Here, 450 μL DPBS pH 7.4, 40 μL of a solution of intermediate F1 (10 mM in DMSO), and 10 μL of a solution of recombinant microbial (bacterial) transglutaminase (Product number T001 from Zedira GmbH, Darmstadt, Germany) (100 U/mL) were added to 500 μL of a solution of the antibody TPP-2090-HC-N297Q (2 mg/mL). The reaction mixture was incubated at 37° C. for 24 hours and afterwards directly analyzed for drug antibody ratio without further purification.

Protein concentration in the reaction mixture: 1 mg/ml

Drug/mAb ratio: 3.5

Example 2A

50 μL DPBS pH 7.4, 40 μL of a solution of intermediate F2 (10 mM in DMSO), and 10 μL of a solution of recombinant microbial (bacterial) transglutaminase (Product number T001 from Zedira GmbH, Darmstadt, Germany) (100 U/mL) were added to 500 μL of a solution of the antibody TPP-2090-HC-N297A (2 mg/mL). The reaction mixture was incubated at 37° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and the transglutaminase from the ADC, and finally concentrated using Amicon Ultracel-30K centrifugation devices (Millipore).

Protein concentration: 1.26 mg/ml

Drug/mAb ratio: 1.6

Example 3A

Here, 450 μL DPBS pH 7.4, 40 μL of a solution of intermediate F3 (10 mM in DMSO), and 10 μL of a solution of recombinant microbial (bacterial) transglutaminase (Product number T001 from Zedira GmbH, Darmstadt, Germany) (100 U/mL) were added to 500 μL of a solution of the antibody TPP-2090-HC-N297A (2 mg/mL). The reaction mixture was incubated at 37° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and the transglutaminase from the ADC, and finally concentrated using Amicon Ultracel-30K centrifugation devices (Millipore).

Protein concentration: 1.06 mg/ml

Drug/mAb ratio: 2.0

Example 4A

Here, 900 μL DPBS pH 7.4, 80 μL of a solution of intermediate F4 (10 mM in DMSO), and 20 μL of a solution of recombinant microbial (bacterial) transglutaminase (Product number T001 from Zedira GmbH, Darmstadt, Germany) (100 U/mL) were added to 1000 μL of a solution of the antibody TPP-2090-HC-N297A (2 mg/mL). The reaction mixture was incubated at 37° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and the transglutaminase from the ADC, and finally concentrated using Amicon Ultracel-30K centrifugation devices (Millipore).

Protein concentration: 2.18 mg/ml

Drug/mAb ratio: 1.7

Example 5A

Here, 900 μL DPBS pH 7.4, 80 μL of a solution of intermediate F5 (10 mM in DMSO), and 20 μL of a solution of recombinant microbial (bacterial) transglutaminase (Product number T001 from Zedira GmbH, Darmstadt, Germany) (100 U/mL) were added to 1000 μL of a solution of the antibody TPP-2090-HC-N297A (2 mg/mL). The reaction mixture was incubated at 37° C. for 24 hours. The ADC was purified by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4 to remove small molecules and the transglutaminase from the ADC, and finally concentrated using Amicon Ultracel-30K centrifugation devices (Millipore).

Protein concentration: 2.24 mg/ml

Drug/mAb ratio: 1.9

Example 6A

Here, 900 μL DPBS pH 7.4, 80 μL of a solution of intermediate F6 (10 mM in DMSO), and 20 μL of a solution of recombinant microbial (bacterial) transglutaminase (Product number T001 from Zedira GmbH, Darmstadt, Germany) (100 U/mL) were added to 1000 μL of a solution of the antibody TPP-2090-HC-N297A (2/mg/mL). The reaction mixture was incubated at 37° C. for 24 hours and afterwards directly analyzed for drug antibody ratio without further purification.

Protein concentration in the reaction mixture: 2.22 mg/ml

Drug/mAb ratio: 2.0

The following examples were synthesized following general procedure A or B for transglutaminase coupling in 5 mg scale and following procedures C or D in larger scale (see chapter B-4):

Example 2A

Precursor: F2, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.66 mg/ml

Drug/mAb ratio: 1.7

Example 3A

Precursor: F3, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.71 mg/ml

Drug/mAb ratio: 1.8

Example 4A

Precursor: F4, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.71 mg/ml

Drug/mAb ratio: 1.8

Example 4A4

Precursor: F4, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.49 mg/ml

Drug/mAb ratio: 3.4

Example 5A

Precursor: F5, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.35 mg/ml

Drug/mAb ratio: 2.0

Coupling of this ADC was also performed in 30 mg scale following the general procedure C in chapter B4:

Protein concentration: 11.2 mg/ml

Drug/mAb ratio: 2.0

Example 5A4

Precursor: F5, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 0.83 mg/ml

Drug/mAb ratio: 3.6

Coupling of this ADC was also performed in 30 mg scale following the general procedure D in chapter B4:

Protein concentration: 10.0 mg/ml

Drug/mAb ratio: 3.8

Example 5E

Precursor: F5, general procedure A, Trastuzumab-HC-N297A (equal to TPP-7510)

Protein concentration: 2.44 mg/ml

Drug/mAb ratio: 1.6

Example 5E4

Precursor: F5, general procedure B, Trastuzumab-HC-N297Q (equal to TPP-7511)

Protein concentration: 2.85 mg/ml

Drug/mAb ratio: 3.6

Example 6A

Precursor: F6, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.63 mg/ml

Drug/mAb ratio: 1.8

Coupling of this ADC was also performed in 30 mg scale following the general procedure C in chapter B4:

Protein concentration: 13.2 mg/ml

Drug/mAb ratio: 1.9

Example 6A4

Precursor: F6, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.63 mg/ml

Drug/mAb ratio: 4.1

Coupling of this ADC was also performed in 30 mg scale following the general procedure D in chapter B4:

Protein concentration: 11.8 mg/ml

Drug/mAb ratio: 3.7

Example 7A

Precursor: F7, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.66 mg/ml

Drug/mAb ratio: 1.9

Example 8A

Precursor: F8, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.68 mg/ml

Drug/mAb ratio: 1.9

Example 9A

Precursor: F9, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.8 mg/ml

Drug/mAb ratio: 2.0

Example 10A

Precursor: F10, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.73 mg/ml

Drug/mAb ratio: 1.8

Example 10A4

Precursor: F10, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.76 mg/ml

Drug/mAb ratio: 3.9

Example 10E4

Precursor: F10, general procedure B, Trastuzumab-HC-N297Q (equal to TPP-7511)

Protein concentration: 1.56 mg/ml

Drug/mAb ratio: 3.9

Example 11A

Precursor: F11, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.82 mg/ml

Drug/mAb ratio: 1.9

Example 11A4

Precursor: F11, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.1 mg/ml

Drug/mAb ratio: 3.6

Example 11E4

Precursor: F11, general procedure B, Trastuzumab-HC-N297Q (equal to TPP-7511).

Protein concentration: 1.75 mg/ml

Drug/mAb ratio: 3.8

Example 12A

Precursor: F12, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.77 mg/ml

Drug/mAb ratio: 1.8

Example 13A

Precursor: F13, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 2.11 mg/ml

Drug/mAb ratio: 1.8

Coupling of this ADC was also performed in 30 mg scale following the general procedure C in chapter B4:

Protein concentration: 11.88 mg/ml

Drug/mAb ratio: 2.0

Example 13A4

Precursor: F13, general procedure D, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Coupling of this ADC was performed in 30 mg scale following the general procedure C in chapter B4:

Protein concentration: 12.0 mg/ml

Drug/mAb ratio: 3.8

Example 14A

Precursor: F14, general procedure A, anti-TWEAKR antibody TPP-2658 (equal to TPP-2090-HC-N297A)

Protein concentration: 1.58 mg/ml

Drug/mAb ratio: 1.7

Example 15A4

Precursor: F15, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.05 mg/ml

Drug/mAb ratio: 3.6

Example 16A4

Precursor: F16, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.78 mg/ml

Drug/mAb ratio: 3.8

Example 16E4

Precursor: F16, general procedure B, Trastuzumab-HC-N297Q (equal to TPP-7511)

Protein concentration: 1.82 mg/ml

Drug/mAb ratio: 3.8

Example 17A4

Precursor: F17, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.71 mg/ml

Drug/mAb ratio: 3.8

Example 17E4

Precursor: F17, general procedure B, Trastuzumab-HC-N297Q (equal to TPP-7511)

Protein concentration: 1.90 mg/ml

Drug/mAb ratio: 3.8

Example 18A4

Precursor: F18, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 1.07 mg/ml

Drug/mAb ratio: 3.7

Example 19A4

Precursor: F19, general procedure B, anti-TWEAKR antibody TPP-5442 (equal to TPP-2090-HC-N297Q)

Protein concentration: 0.92 mg/ml

Drug/mAb ratio: 3.3

Metabolite Example M100 N⁶-L-gamma-glutamyl-N²-acetyl-N-[2-({(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]butanoyl}amino)ethyl]-L-lysinamide trifluoro acetate (1:1)

First intermediate C58 was coupled with intermediate L12 in DMF in the presence of HATU and N,N-diisopropylethylamine; subsequently the Teoc-protecting group was cleaved using 4 equivalents of zinc chloride in 2,2,2 trifluoroethanol under heating for 2 h to 50° C. and after addition of 4 equivalents of EDTA the product was purified by HPLC. Finally the Z protecting group and the benzyl ester were cleaved by hydrogenation in methanol over 10% palladium/activated charcoal under normal pressure.

LC-MS (Method 1): R_(t)=0.74 min; MS (ESIpos): m/z=855 (M+H)⁺.

C: Assessment of Biological Efficacy

The biological activity of the compounds according to the invention can be shown in the assays described below:

C-1a Determination of the Cytotoxic Effects of the ADCs Directed Against TWEAKR

The analysis of the cytotoxic effects of the anti-TWEAKR-ADCs was carried out with various cell lines:

NCI-H292: human mucoepidermoid lung carcinoma cells, ATCC-CRL-1848, standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine)+10% FCS (Biochrom; #S0415), TWEAKR-positive, EGFR-positive.

SK-HEP-1: human liver carcinoma cells, ATCC No. HTB-52, standard medium: MEM with Earle's salt+Glutamax I (Invitrogen 41090)+10% heat inactivated FCS (Fa. gibco, No. 10500-064); EGFR-positive, TWEAKR positive.

LoVo: human colorectal carcinoma cells, ATCC No. CCL-229, standard medium: Kaighn's+L-Glutamin (Invitrogen 21127)+10% heat inactivated FCS (Fa. gibco, No. 10500-064), TWEAKR-positive

BxPC3: human pancreas carcinoma cells, ATCC-CRL-1687, standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine)+10% FCS (Biochrom; #S0415), TWEAKR-positive.

KPL4: human breast carcinoma cells, standard medium: RPMI 1640+GlutaMAX I+10% FBS, cell bank, Bayer Pharma AG (identity checked and confirmed on 19 Jul. 2012 at DSMZ), Berlin, ERBB2-positive.

The cells are cultivated by a standard method, as indicated in the American Tissue Type Collection (ATCC) for the respective cell lines.

MTT Assay

The test was carried out by detaching the cells with a solution of Accutase in PBS (Biochrom AG #L2143), pelleting, resuspending in culture medium, counting and seeding the cells into a 96-well culture plate with white bottom (Costar #3610, LoVo:1000 cells/well, SK-HEP-1: 1200 cell/well, NCI H292: 1500 cell/well in a total volume of 100 μL). The cells were then incubated in an incubator at 37° C. and 5% carbon dioxide. After 48 h, the antibody drug conjugates were added in 10 μl of culture medium in concentrations of from 10⁻⁵M to 10⁻¹³M to the cells (triplicates) and incubated in an incubator at 37° C. and 5% carbon dioxide. After 96h, the proliferation was measured using the MTT assay (ATCC, Manassas, Va., USA, catalogue No. 30-1010K). At the end of the selected incubation time, the MTT reagent was added and incubated with the cells for 4h, followed by lysis of the cells overnight by addition of the detergent. The dye formed was detected at 570 nm (Infinite M1000 pro, Fa. Tecan). Based on the measured data the IC₅₀ value was determined from the DRC (dose response curve). The proliferation of cells which were not treated with test substance but were otherwise treated identically was defined as the 100% value. The data derived from a selected example are summarized in Table 1.

TABLE 1 NCI-H292 SK-HEP-1 IC₅₀ [M] IC₅₀ [M] example MTT Assay MTT Assay 1A 1.19E−09 3.35E−09 2A 5.00E−07 7.85E−08 3A 4.34E−09 3.84E−08 4A 5.90E−10 2.26E−10  4A4 2.99E−10 7.88E−11 5A 2.20E−09 1.49E−09  5A4 8.26E−10 1.52E−10 6A 3.98E−10 7.43E−10  6A4 1.07E−10 3.93E−11 7A 5.00E−07 5.00E−07 8A 3.07E−09 1.34E−09 9A 2.09E−09 2.12E−09 10A  8.51E−10 8.87E−10 10A4 1.84E−10 1.71E−10 11A  3.12E−09 6.53E−10 11A4 3.61E−10 3.34E−11 12A  3.18E−10 1.77E−10 13A  1.50E−09 1.92E−09 14A  2.49E−09 3.13E−09 15A4 1.31E−11 3.04E−11 16A4 2.26E−10 8.71E−11 17A4 1.84E−10 1.13E−10 18A4 4.53E−10 1.23E10 19A4 1.90E−10 4.03E−10

TABLE 2 KPL4 IC₅₀ [M] Beispiel MTT Assay 5E   5E−07  5E4 2.74E−10 10E4 3.29E−10 11E4 2.68E−11 16E4 5.21E−11 17E4 3.66E−11 C-1b Determination of the Inhibition of the Kinesin Spindle Protein KSP/Eg5

The motor domain of the human kinesin spindle protein KSP/Eg5 (from tebu-bio/Cytoskeleton Inc, No. 027EG01-XL) is incubated at a concentration of 10 nM with 50 μg/ml taxol- (from Sigma No. T7191-5MG) stabilized microtubuli (bovine or porcine, from tebu-bio/Cytoskeleton Inc) for 5 min at RT in 15 mM PIPES, pH 6.8 (5 mM MgCl₂ and 10 mM DTT, from Sigma). The freshly prepared mixture was aliquoted into a 384-well MTP. The inhibitors to be examined at concentrations of 1.0×10⁻⁶ M to 1.0×10⁻¹³ M and ATP (final concentration 500 μM, from Sigma) were then added. Incubation was carried out at RT for 2 h. ATPase activity was detected by detecting the inorganic phosphate formed using malachite green (from Biomol). After addition of the reagent, the assay was incubated at RT for 50 minutes prior to detection of the absorption at a wavelength of 620 nm. Monastrol (Fa. Sigma, M8515-1 mg) and Ispinesib (from Adooq A10486) were used as positive control. The individual data of the dose-activity curve are octuple determinations. The IC₅₀ values are means of three independent experiments. The 100% control was the sample which had not been treated with inhibitors.

In the following table 2 IC₅₀-values of representative examples derived from the described assay and the corresponding cytotoxicity data (MTT-Assay) are summarized:

TABLE 2 NCI-H292 KPL-4 KSP-Assay IC₅₀ [M] IC₅₀ [M] Beispiele IC₅₀ [M] MTT Assay MTT Assay M100 2.45E−09 >5.00E−07 >5.00E−07 C-2 Internalisation Assay

Internalisation is a key process which enables specific and efficient provision of the cytotoxic payload in antigen-expressing cancer cells via antibody drug conjugates (ADC). This process is monitored via fluorescent labelling of specific TWEAKR antibodies and an isotype control antibody. First, the fluorescent dye is conjugated to lysines of the antibody. Conjugation is carried out using a two-fold molar excess of CypHer 5E mono NHS ester (Batch 357392, GE Healthcare) at pH 8.3. After the coupling, the reaction mixture was purified using gelchromatography (Zeba Spin Desalting Columns, 40K, Fa. Thermo Scientific, No. 87768; Elutionspuffer: DULBECCO'S PBS, Fa. Sima-Aldrich, No. D8537), to remove excess dye and to adjust the pH. The protein solution was then concentrated (VIVASPIN 500, from Sartorius stedim biotec). Determination of the dye load of the antibody was by spectrophotometric analysis (NanoDrop) and subsequent calculation (D: P=A_(dye) ε_(protein): (A₂₈₀-0.16 A_(dye))ε_(dye)). The dye load of the TWEAKR antibody examined here and the isotype control were of a comparable order. In cell binding assays, it was confirmed that the conjugation did not lead to a change in the affinity of the antibody.

The labelled antibodies were used for the internalisation assay. Prior to the start of the treatment, the cells (2×10⁴/well) were seeded in 100 μL medium in a 96-well MTP (fat, black, clear bottom No 4308776, from Applied Biosystems). After 18 h of incubation at 37° C./5% CO₂, the medium was replaced and labelled anti-TWEAKR antibodies were added in different concentrations (10, 5, 2.5, 1, 0.1 μg/mL). The same treatment protocol was applied to the labelled isotype control (negative control). The chosen incubation times are 0h, 0.25h, 0.5h, 1h, 1.5h, 2h, 3h, 6h and 24h. The fluorescence measurement was carried out using the InCellanalyser 1000 (from GE Healthcare). This was followed by kinetic evaluation via measurement of the parameters granule counts/cell and total granule intensity/cell.

Following binding to the TWEAKR, TWEAKR antibodies were examined for the internalisation ability. For this purpose, cells with different TWEAKR expression levels were chosen. A target-mediated specific internalisation was observed with the TWEAKR antibodies, whereas the isotype control showed no internalisation.

The commercially available antibodies (Cetuximab, Nimotuzumab, Herceptin) were treated identical and internalization was performed as described above using the respective Target-expressing cells. For all antibodies a target-dependent internalization could be observed while the isotype control showed no internalization.

C-3 In Vitro Tests for Determining Cell Permeability

The cell permeability of a substance can be investigated by means of in vitro testing in a flux assay using Caco-2 cells [M. D. Troutman and D. R. Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. For this purpose, the cells were cultured for 15-16 days on 24-well filter plates. For the determination of permeation, the respective working example was applied in a HEPES buffer to the cells either apically (A) or basally (B) and incubated for 2 hours. After 0 hours and after 2 hours, samples were taken from the cis and trans compartments. The samples were separated by HPLC (Agilent 1200, Böblingen, Germany) using reverse phase columns. The HPLC system was coupled via a Turbo Ion Spray Interface to a Triple Quadropol mass spectrometer API 4000 (Applied Biosystems Applera, Darmstadt, Germany). The permeability was evaluated on the basis of a Papp value, which was calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. A substance was classified as actively transported when the ratio of P_(app) (B−A) to P_(app) (A−B) (efflux ratio) was >2 or <0.5.

Of critical importance for toxophores which are released intracellularly is the permeability from B to A [P_(app) (B−A)] and the ratio of P_(app) (B−A) to P_(app) (A−B) (efflux ratio): the lower this permeability, the slower the active and passive transport processes of the substance through the monolayer of Caco-2 cells. If additionally the efflux ratio does not indicate any active transport, the substance may, following intracellular release, remain longer in the cell. Hence, there is also more time available for interaction with the biochemical target (in this case: kinesin spindle protein, KSP/Eg5).

TABLE 3 P_(app) (B-A) Metabolite example [nm/s] Efflux ratio M100 1.2 0.7 C-4 In Vitro Tests for Determining the Substrate Properties for P-Glycoprotein (P-Gp)

Many tumour cells express transporter proteins for drugs, and this frequently accompanies the development of resistance towards cytostatics. Substances which are not substrates of such transporter proteins, such as P-glycoprotein (P-gp) or BCRP, for example, could therefore exhibit an improved activity profile.

The substrate properties of a substance for P-gp (ABCB1) were determined by means of a flux assay using LLC-PK1 cells which overexpress P-gp (L-MDR1 cells) [A. H. Schinkel et al., J. Clin. Invest. 96, 1698-1705 (1995)]. For this purpose, the LLC-PK1 cells or L MDR1 cells were cultured on 96-well filter plates for 3-4 days. For determination of the permeation, the respective test substance, alone or in the presence of an inhibitor (such as ivermectin or verapamil, for example), was applied in a HEPES buffer to the cells either apically (A) or basally (B) and incubated for 2 hours. After 0 hours and after 2 hours, samples were taken from the cis and trans compartments. The samples were separated by HPLC using reverse phase columns. The HPLC system was coupled via a Turbo Ion Spray Interface to a Triple Quadropol mass spectrometer API 3000 (Applied Biosystems Applera, Darmstadt, Germany). The permeability was evaluated on the basis of a Papp value, which was calculated using the formula published by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. A substance was classified as P=gp substrate when the efflux ratio of P_(app) (B−A) to P_(app) (A−B) was >2.

As further criteria for the evaluation of the P-gp substrate properties, the efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in the presence or absence of an inhibitor may be compared. If these values differ by a factor of more than 2, the substance in question is a P-gp substrate.

C-5 Pharmakokinetics

C5a: Identification of the ADC Metabolites after Internalisation In Vitro

Description of the Method:

Internalisation studies with immunoconjugates are carried out to analyse metabolites formed intracellularly. To this end, human lung tumour cells NCI H292 (3×10⁵/well) are sown in 6-well plates and incubated overnight (37° C., 5% CO₂). The cells are treated with 10 μg/ml of the ADC to be examined. Internalisation is carried out at 37° C. and 5% CO₂. At various time points (0, 4, 24, 48, 72 h), cell samples are taken for further analysis. First, the supernatants (about 5 ml) are harvested and, after centrifugation (2 min, RT, 1000 rpm Heraeus Variofuge 3.0R), stored at −80° C. The cells are washed with PBS and detached with Accutase, and the cell number is determined. After another washing, a defined number of cells (2×10⁵) is treated with 100 μl of lysis buffer (Mammalian Cell Lysis Kit (Sigma MCL1) and incubated with continuous shaking (Thermomixer, 15 min, 4° C., 650 rpm) in Protein LoBind tubes (eppendorf Cat. No. 0030 108.116). After the incubation, the lysate is centrifuged (10 min, 4° C., 12000 g, eppendorf 5415R) and the supernatant is harvested. The supernatant obtained is stored at −80° C. All samples are then analysed as follows.

Measurement of the compounds in the culture supernatant or cell lysate is carried out after precipitation of the proteins with methanol or acetonitrile by high-pressure liquid chromatography (HPLC) coupled to a triple-quadrupole mass spectrometer (MS).

For work-up of 50 μl of culture supernatant/cell lysate, 150 μl of precipitation reagent (generally acetonitrile) are added and the mixture is shaken for 10 seconds. The precipitation reagent contains an internal standard (ISTD) in a suitable concentration (generally in the range of 20-100 ng/ml). After 3 minutes of centrifugation at 16000 g, the supernatant is transferred into an autosampler vial, made up with 500 μl of a buffer suitable for the mobile phase and shaken again.

The two matrix samples are then measured using the HPLC-coupled triple-quadrupol mass spectrometer API6500 from AB SCIEX Deutschland GmbH.

For calibration, concentrations of 0.5-2000 μg/l are added to plasma samples. The detection limit (LOQ) is about 2 μgl. The linear range extends from 2 to 1000 μg/l.

For calibration of the tumour samples, concentrations of 0.5-200 μg/l are added to the supernatant of untreated tumours. The detection limit is 4 μg/l. The linear range extends from 4 to 200 μg/l.

Quality controls for testing validity contain 5 and 50 μg/l.

C5b: Identification of the ADC Metabolites In Vivo

After i.v. administration of 3-30 mg/kg of different ADCs, the plasma and tumour concentrations of the ADCs and any metabolites occurring can be measured, and the pharmacokinetic parameters such as clearance (CL), area under the curve (AUC) and half-times (t_(1/2)) can be calculated.

Analysis for Quantification of any Metabolites Occurring

Measurement of the compounds in plasma and tumour is carried out after precipitation of the proteins with methanol or acetonitrile by high-pressure liquid chromatography (HPLC) coupled to a triple-quadrupole mass spectrometer (MS).

For work-up of 50 μl of plasma, 250 μl of precipitation reagent (generally acetonitrile) are added and the mixture is shaken for 10 seconds. The precipitation reagent contains an internal standard (ISTD) in a suitable concentration (generally in the range of 20-100 ng/ml). After 3 minutes of centrifugation at 16000 g, the supernatant is transferred into an autosampler vial, made up with 500 μl of a buffer suitable for the mobile phase and shaken again.

During the work-up of a tumour, the latter is treated with 3 times the amount of extraction buffer. The extraction buffer contains 50 ml of Tissue Protein Extraction Reagent (Pierce, Rockford, Ill.), two pellets of Complete-Protease-Inhibitor-Cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethylsulphonyl fluoride (Sigma, St. Louis, Mo.) in a final concentration of 1 mM. The sample is homogenized twice for 20 minutes in a Tissuelyser II (Qiagen), at maximum stroke number. 50 μl of the homogenate are transferred into an autosampler vial and made up with 150 μl of methanol including ISTD. After 3 minutes of centrifugation at 16000 g, 10 μl of the supernatant are made up with 180 μl of a buffer suitable for the mobile phase and shaken again. The tumour sample is then ready for measuring.

The two matrix samples are then measured using the HPLC-coupled triple-quadrupol mass spectrometer API6500 from AB SCIEX Deutschland GmbH.

For calibration, concentrations of 0.5-2000 μg/l are added to plasma samples. The detection limit (LOQ) is about 2 μg/l. The linear range extends from 2 to 1000 μg/l.

For calibration of the tumour samples, concentrations of 0.5-2000 μg/l are added to the supernatant of untreated tumours. The detection limit is 5 μg/l. The linear range extends from 5 to 200 μg/l.

Quality controls for testing validity contain 5 and 50 μg/l, in plasma additionally 500 μg/l.

Analysis for Quantification of the Antibodies Used

The antibody part of the ADCs was determined using a ligand binding assay (ELISA) as total IgG concentration in plasma samples and tumour lysates. Here, the sandwich ELISA format was used. This ELISA had been qualified and validated for the determination in plasma and tumour samples. The ELISA plates were coated with anti-human goat IgG Fc antibodies. After incubation with the sample, the plates were washed and incubated with a detector conjugate of simian anti-human IgG(H+L) antibody and horseradish peroxidase (HRP). After a further washing step, the HRP substrate was added to OPD and the colour development was monitored via absorption at 490 nm. Standard samples having a known IgG concentration were fitted using a 4-parameter equation. Within the lower (LLOQ) and upper (ULOQ) quantification limits, the unknown concentrations were determined by interpolation.

C-6 Activity Test In Vivo

The activity of the conjugates according to the invention was tested, for example, using xenograft models. The person skilled in the art is familiar with methods in the prior art which allow the activity of the compounds according to the invention to be tested (see, for example, WO 2005/081711; Polson et al., Cancer Res. 2009 Mar. 15; 69(6):2358-64). To this end, a tumour cell line expressing the target molecule of the binder was implanted into rodents (for example mice). A conjugate according to the invention, an isotype control conjugate, a control antibody or isotonic saline was then administered to the implant animals. The administration took place once or more than once. Following an incubation time of several days, the size of the tumour was determined by comparing conjugate-treated animals and the control group. The conjugate-treated animals displayed a smaller tumour size.

Growth Inhibition/Regression of Experimental Tumours in the Mouse

Human tumour cells expressing the antigen for the antibody drug conjugate are inoculated subcutaneously into the flank of immunosuppressed mice, for example NMRi nude or SCID mice. 1-10 million cells are detached from the cell culture, centrifuged and resuspended in medium or medium/matrigel. The cell suspension is injected under the skin of the mouse.

Within a few days, a tumour grows. Treatment is commenced after the tumour is established, at a tumour size of approximately 40 mm². To examine the effect on larger tumours, treatment may be initiated only at a tumour size of 50-100 mm².

Treatment with ADCs is carried out via the intravenous route into the tail vein of the mouse. The ADC is administered in a volume of 5 ml/kg.

The treatment protocol depends on the pharmacokinetics of the antibody. As standard, treatment takes place three times in succession every fourth day. For a quick assessment, a protocol with a single treatment may be employed. However, the treatment may also be continued, or a second cycle of three treatment days may follow at a later time.

As standard, 8 animals are used per treatment group. In addition to the groups to which the active substances are administered, one group is treated as control group only with the buffer, according to the same protocol.

During the experiment, the tumour area is measured regularly in two dimensions (length/width) using a caliper. The tumour area is determined as length×width. The ratio of the mean tumour area of the treatment group to that of the control group is stated as T/C area.

When after the end of the treatment all groups of the experiment are terminated at the same time, the tumours can be removed and weighed. The ratio of the mean tumour weights of the treatment group to that of the control group is stated as T/C weight.

D. Working Examples of Pharmaceutical Compositions

The compounds according to the invention can be converted to pharmaceutical formulations as follows:

i.v. Solution:

The compound according to the invention is dissolved in a concentration below the saturation solubility in a physiologically acceptable solvent (e.g. isotonic saline solution, D-PBS, or a formulation with glycine and sodium chloride in citrate buffer with addition of polysorbate 80). The solution is subjected to sterile filtration and dispensed into sterile and pyrogen-free injection vessels.

i.v. Solution:

The compounds according to the invention can be converted to the administration forms mentioned. This can be accomplished in a manner known per se by “mixing with” or “dissolving in” inert, non-toxic, pharmaceutically suitable excipients (e.g. buffer substances, stabilizers, solubilizers, preservatives). The following, for example, may be present: amino acids (glycine, histidine, methionine, arginine, lysine, leucine, isoleucine, threonine, glutamic acid, phenylalanine and others), sugars and related compounds (glucose, saccharose, mannitol, trehalose, sucrose, mannose, lactose, sorbitol), glycerol, sodium salts, potassium, ammonium salts and calcium salts (e.g. sodium chloride, potassium chloride or disodiumhydrogenphosphate and many others), acetate/acetic acid buffer systems, phosphate buffer systems, citric acid and citrate buffer systems, trometamol (TRIS and TRIS salts), Polysorbates (e.g. Polysorbate 80 and Polysorbate 20), Poloxamers (e.g. Poloxamer 188 and Poloxamer 171), Macrogols (PEG derivatives, e.g. 3350), Triton X-100, EDTA salts, glutathione, albumins (e.g. human), urea, benzyl alcohol, phenol, chlorocresol, metacresol, benzalkonium chloride and many others.

Lyophilizate for Subsequent Conversion into an i.v., s.c. Or i.m. Solution:

Alternatively the compounds of the invention may be converted into a stable lyophilizate (possibly with the aid of abovementioned excipients) and, before being administered, reconstituted with a suitable solvent (e.g. injection-grade water, isotonic saline solution) and administered.

Working Examples of Anti-TWEAKR Antibodies

All examples were carried out using standard methods known to the person skilled in the art, unless described here in detail. Routine methods of molecular biology of the examples that follow can be carried out as described in standard laboratory textbooks such as Sambrook et al., Molecular Cloning: a Laboratory Manual, 2. Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

AK Example 1: Antibody Preparation Using an Antibody Library

A complete human phage display library (Hoet R M et al, Nat Biotechnol 2005; 23(3):344-8) was employed to isolate TWEAKR-specific human monoclonal antibodies of the present invention by protein panning (Hoogenboom H. R., Nat Biotechnol 2005; 23(3):1105-16), where dimeric Fc-fused extracellular domains of human and murine TWEAKR were immobilized as target.

TABLE AK-1 List of recombinant antigens used for antibody selection Nomenclature Description SEQ ID NO TPP-599 HUMAN-TNFRSF12Aaa28-80-hIgG1-Fc 138 TPP-601 MURIN-TNFRSF12Aaa28-80-hIgG1-Fc 137

The antigens were biotinylated using an about 2-fold molar excess of biotin-LC-NHS (Pierce; Cat. No. 21347) according to the instructions of the manufacturer and desalted using Zeba desalting columns (Pierce; Cat. No. 89889). Washed magnetic beads (DynaBeads) were incubated overnight with 200 nM biotinylated antigen at 4° C. and blocked for 1 h at 4° C. with blocking buffer (PBS with 3% BSA, 0.05% Tween-20). The blocked Fab phage library was added to the blocked TWEAKR beads (DynaBeads Streptavidin-M280-Invitrogen 112-06D) and incubated at room temperature for 30 min. After stringent washing (3× with blocking buffer and 9× with PBS (150 mM NaCl; 8 mM Na2HPO4; 1.5 mM KH2PO4; adjusted to pH=7.4-7.6) with 0.05% Tween-20), Fab phages binding specifically to biotinylated TWEAKR beads (DynaBeads Streptavidin-M280-Invitrogen 112-06D) were resuspended in PBS and, for amplification, used directly for infecting Escherichia coli strain TG1. In the second selection round, two murine TWEAKR (200 nM) were used to select for cross-reactive binders, and in the third selection round the concentration of human TWEAKR was reduced (100 nM) to increase the selection pressure for high-affinity binders.

11 different Fab phages were identified and the corresponding antibodies were cloned into a mammalian IgG expression vector which provided the missing CH2-CH3 domains not present in the soluble Fab. The resulting IgGs were expressed transiently in mammalian cells as described in Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007. Briefly, a CMV promoter-based expression plasmid was transfected into HEK293-6E cells and incubated in Fernbach bottles or Wave bags. Expression took place at 37° C. for 5 to 6 days in F17 medium (Invitrogen). 1% Ultra-Low IgG FCS (Invitrogen) and 0.5 mM valproic acid (Sigma) were added as supplements 24 h after the transfection. The antibodies were purified by protein-A chromatography and characterized further by their binding affinity to soluble monomeric TWEAKR using ELISA and BIAcore analysis, as described in AK-Example 2.

TABLE AK-2 List of recombinant antigen used for the affinity measurement Cat. No. SEQ Nomenclature Description Origin (Fitzgerald Inc) ID NO TPP-2305 hTNFRSF12 amino human 30R-AT080 168 acids a28-80

To determine the cell binding characteristics of the anti-TWEAKR antibodies, binding to a number of cell lines (HT29, HS68, HS578) was examined by flow cytometry. The cells were suspended in dilutions of the antibodies (5 μg/ml) in FACS buffer and incubated on ice for 1 h. A second antibody (PE goat-anti-human IgG, Dianova #109-115-098) was then added. After 1 h of incubation on ice, the cells were analysed by flow cytometry using an FACS array (BD Biosciences).

NF-kappaB reporter gene assays were carried out to assess the agonistic activity of all 11 antibodies identified (human IgG1). HEK293 cells were transiently transfected with an NF-kappaB reporter construct (BioCat, Cat. No. LR-0051-PA) using 293fectin according to the instructions of the manufacturer. Transfected cells were sown in F17 media (serum-free; Invitrogen) at 37 C, 5% CO2 into white polylysine-coated 384-well plates (BD). The next day, the cells were stimulated with various concentrations of purified antibodies for 6 h, and a luciferase assay was then carried out using standard methods.

Internalisation was monitored via fluorescence labelling of anti-TWEAKR antibodies (CypHer 5E mono NHS ester; GE Healthcare). Prior to the treatment, HT29 cells were sown (2×10⁴/well) in 100 μl of medium in 96-well MTP plates (thick, black, transparent botton, No. 4308776, Applied Biosystems). After 18 h of incubation at 37° C./5% CO₂, the medium was replaced and labelled anti-TWEAKR antibodies were added in different concentrations (10, 5, 2.5, 1, 0.1 μg/ml). The chosen incubation time was 0, 0.25, 0.5, 1, 1.5, 2, 3, 6 and 24h. Fluorescence measurement was carried out in an InCell-analyser 1000 (GE Healthcare).

The antibody having the highest in vitro activity (TPP-883) was selected for further activity and affinity maturation.

TPP-883 SEQ ID NO. 71 AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSS PGITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 72 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWV SYISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARGGDGYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO.71) and heavy (SEQ ID NO.72) chains of TPP-883; CDRs both of the heavy and the light chain are underlined.

Maturation was carried out in a first mutations collection round, followed by recombination of those amino acid modifications which increased affinity and activity most. For collecting mutations NNK (N=AGCT, K=G or T), randomization was carried out at the following individual amino acid positions by site-directed mutagenesis using synthetic oligonucleotides including NNK codon diversification (continuous amino acid nomenclature): S35, S36, Y37 and N39 in CDR-L1; A51, S53, S54, Q56 and S57 in CDR-L2; S92, Y93, S94, S95, G97 and I98 in CDR-L3; P31, Y32, P33, M34 and M35 in CDR-H1; Y50, S52, P53, S54, G56, K57 and H59 in CDR-H2; G99, G100, D101, G102, Y103, F104, D105 and Y106 in CDR-H3. The DNA of all individual NNK saturation mutagenesis libraries was cloned into a mammalian IgG expression vector for activity maturation or into a phagemid vector for affinity maturation. Affinity maturation was carried out by phage panning. Washed magnetic beads (DynaBeads) were incubated overnight with 10 nM, 1 nM, 100 pM or 10 pM biotinylated antigen at 4° C. and blocked for 1 h at 4° C. with blocking buffer (PBS with 3% BSA, 0.05% Tween-20). The blocked Fab phage library was added in 10000-fold, 1000-fold or 100-fold excess, compared to the theoretical library complexity, to the blocked TWEAKR-DynaBeads and incubated at room temperature for 30 min. That means that 12 strategies were followed in total (4 antigen concentrations×3 Fab phage titres). After stringent washing (3× with blocking buffer and 9× with PBS with 0.05% Tween-20), Fab phages binding specifically to biotinylated TWEAKR DynaBeads (DynaBeads Streptavidin-M280-Invitrogen 112-06D) were resuspended in PBS and, for amplification, used directly for infecting Escherichia coli strain TG1. In selection round two, the concentration of human TWEAKR-Fc was reduced (1 nM, 100 pM, 10 pM and 1 pM), and the same Fab phage titre was used for all 12 strategies (4.4×10¹¹). For the expression of soluble Fab, the phagemid vector was digested with MluI to remove the gene-III membrane anchor sequence required for the Fab display on the phage, and the vector was re-ligated. 96 variants of each of the 12 selection pools were expressed as soluble Fabs and examined in an ELISA format. To this end, 2.5 nM biotinylated TWEAKR-Fc were antigen-coated, and binding of soluble Fabs was demonstrated using anti-c-Myc antibodies (Abeam ab62928). 7 single substitution variants (consecutive amino acid nomenclature) with improved binding to TWEAKR-Fc (Seq ID No 138) were demonstrated: S36G of CDR-L1, A51Q and S57K of CDR-L2, S94T and G97F of CDR-L3, M351 of CDR-H1 and G102T of CDR-H3. For the activity maturation, HEK293 cells were transfected with an NF-kappaB reporter (BioCat, Cat. No. LR-0051-PA). Transfected cells were sown in F17 media (serum-free; Invitrogen) in white, polylysine-coated 384-well plates (BD), and individual variants of the NNK-diversified position antibodies (human IgG1) libraries were expressed transiently in mammalian cells. The next day, NF-kappaB reporter cells were stimulated with the individual NNK antibody variants expressed for 6 h, and a luciferase assay was then carried out using standard methods. 1 single substitution variant having improved agonistic activity was detected: G102T of CDR-H3. This variant was also obtained by affinity maturation, and there, too, it showed the highest enhancement of affinity. After mutation collection by affinity and activity screening, all 7 favourable individual substitutions (library complexity: 128 variants) were recombined into a recombination library. To this end, oligonucleotides were synthesized to introduce selected mutations or the corresponding wild type amino acid at each selected position. The library was established using successive rounds of overlap extension PCR. The final PCR product was ligated into a bacterial soluble Fab expression vector, and 528 variants were selected at random (˜4-fold excess of the sample taken) for an equilibrium ELISA screen with soluble Fabs, as described above. In the end, 7 variants were selected based on increased affinity compared to the best single substitution variant G102T. The corresponding DNA of these was cloned into a mammalian IgG expression vector and examined for functional activity in the above-mentioned NF-kappaB reporter cell assay. Finally, the sequences obtained were compared with human germ line sequences, and deviations without any significant effect on the affinity and the efficacy were adapted. Antibodies having the sequences below were obtained by antibody library screening and by affinity and/or activity maturation:

TPP-2090 SEQ ID NO. 1: DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLI YQASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPF ITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 2: EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEWV SYISPSGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG

Amino acid sequences of the light (SEQ ID NO.1) and heavy (SEQ ID NO.2) chains of TPP-2090;

CDRs both of the heavy and the light chain are underlined. Based on the sequence of TPP-2090, the aglycosylated antibodies TPP-2090-HC-N297A (comprising the mutation in the heavy chain N297A (Kabat, EU numbering) and TPP-2090-HC-N-297Q (comprising the mutation in the heavy chain N297Q (Kabat, EU numbering) were generated by site directed mutagenesis:

TPP-2090-HC-N297A Light chain (SEQ ID NO: 1): DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLL IYQASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTS PFITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain (SEQ ID NO.241): EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEW VSYISPSGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG TPP-2090-HC-N297Q Light chain (SEQ ID NO. 1): DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLL IYQASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTS PFITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain (SEQ ID NO. 242): EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEW VSYISPSGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG TPP-2149 SEQ ID NO. 11 DIQMTQSPATLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLL IYQASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTS PFITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 12 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEW VSYISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO.11) and heavy (SEQ ID NO.12) chains of TPP-2149; CDRs both of the heavy and the light chain are underlined.

TPP-2093 SEQ ID NO. 21 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYQASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTS PFITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 22 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEW VSYISPSGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO.21) and heavy (SEQ ID NO.22) chains of TPP-2093; CDRs both of the heavy and the light chain are underlined.

TPP-2148 SEQ ID NO. 31 DIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYQASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTS PPFITFGPGTKVEIKRTVAAPSVFIFPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 32 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEW VSYISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO.31) and heavy (SEQ ID NO.32) chains of TPP-2148; CDRs both of the heavy and the light chain are underlined.

TPP-2084 SEQ ID NO. 41 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYST PGITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 42 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEW VSYISPSGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO.41) and heavy (SEQ ID NO.42) chains of TPP-2084; CDRs both of the heavy and the light chain are underlined.

TPP-2077 SEQ ID NO. 51 DIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSS PGITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKLYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 52 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEW VSYISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequences of the light (SEQ ID NO.51) and heavy (SEQ ID NO.52) chains of TPP-2077; CDRs both of the heavy and the light chain are underlined.

TPP-1538 SEQ ID NO. 61 AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPG ITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 62 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVS YISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG Amino acid sequences of the light (SEQ ID NO.61) and heavy (SEQ ID NO.62) chains of TPP-1538; CDRs both of the heavy and the light chain are underlined.

TPP-1854 SEQ ID NO. 81 AQDIQMTQSPATLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLL IYNASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPF ITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 82 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMIWVRQAPGKGLEWVS YISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG Amino acid sequences of the light (SEQ ID NO.81) and heavy (SEQ ID NO.82) chains of TPP-1854; CDRs both of the heavy and the light chain are underlined.

TPP-1853 SEQ ID NO. 91 AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYNASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPG ITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 92 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVS YISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG Amino acid sequences of the light (SEQ ID NO.91) and heavy (SEQ ID NO.92) chains of TPP-1853; CDRs both of the heavy and the light chain are underlined.

TPP-1857 SEQ ID NO. 101 AQDIQMTQSPATLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLL IYNASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPG ITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 102 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVS YISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG Amino acid sequences of the light (SEQ ID NO.101) and heavy (SEQ ID NO.102) chains of TPP-1857; CDRs both of the heavy and the light chain are underlined.

TPP-1858 SEQ ID NO. 111 AQDIQMTQSPATLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLL IYNASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTSPF ITFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 112 EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYPMMWVRQAPGKGLEWVS YISPSGGKTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GGDTYFDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG Amino acid sequences of the light (SEQ ID NO.111) and heavy (SEQ ID NO.112) chains of TPP-1858; CDRs both of the heavy and the light chain are underlined.

Example 2: Biochemical Characteristics of the Antibodies

Determination of Binding Affinities by Biacore Analysis:

Binding affinities of anti-TWEAKR antibodies were examined using surface plasmon resonance analysis on a Biacore T100 instrument (GE Healthcare Biacore, Inc.). The antibodies were immobilized on a CMS sensor chip using an indirect capture reagent, anti-human IgG(Fc). Reagents of the “Human Antibody Capture Kit” (BR-1008-39, GE Healthcare Biacore, Inc.) were used as described by the manufacturer. Anti-TWEAKR antibodies were injected at a concentration of 10 μg/ml at 10 μl/min for 10 sec.

TABLE AK-3 List of recombinant antigen (TWEAKR) used for affinity measurement Cat. No. SEQ Nomenclature Description Origin (Fitzgerald Inc) ID NO TPP-2305 hTNFRSF12 amino human 30R-AT080 168 acids a28-80

TABLE AK-4 List of antibodies used for the affinity measurement SEQ ID NO Nomenclature Description Light chain Heavy chain P3G5(TPP-2195) murine IgG2a 121 122 P4A8(TPP-1324) human IgG1 125 126 P2D3(TPP-2196) murine IgG2a 131 132 136.1(TPP-2194) murine IgG2a 123 124 PDL-192(TPP-1104) human IgG1 127 128 18.3.3(TPP-2193) murine IgG2a 129 130 TPP-883 human IgG1 71 72 TPP-1538 human IgG1 61 62 TPP-2077 human IgG1 51 52 TPP-2084 human IgG1 41 42 TPP-2148 human IgG1 31 32 TPP-2093 human IgG1 21 22 TPP-2149 human IgG1 11 12 TPP-2090 human IgG1 1 2

TABLE AK-5 List of commercially available antibodies used for the affinity measurement Nomenclature Description Cat. No. (Abcam) ITEM-1 murine IgG1 ab21359 ITEM-4 murine IgG1 ab21127

Various concentrations (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, 1.56 nM) of purified recombinant human TWEAKR protein (TPP-2305, SEQ ID NO:168) in HEPES-EP buffer (GE Healthcare Biacore, Inc.) were injected over immobilised anti-TWEAKR antibodies at a flow rate of 60 μl/min for 3 minutes, the dissociation time being 5 minutes. Sensorgrams were generated after in-line reference cell correction, followed by subtraction of the buffer sample. The dissociation constant (K_(D)) was calculated based on the ratio of association (k_(on)) and dissociation (k_(off)) constants, obtained by fitting sensorgrams using a 1:1 first order binding model.

TABLE AK-6 Monovalent K_(D) values of anti-TWEAKR antibodies measured using Biacore with TWEAKR protein (TPP-2305 (SEQ ID NO: 168)). ka (1/Ms) kd (1/s) K_(D) (nM) TPP-883 4.40E+06 9.10E−01 205.9 TPP-1538 4.20E+06 1.10E−01 27.6 TPP-2077 3.00E+06 8.60E−02 28.9 TPP-2084 4.20E+06 1.10E−01 27.6 TPP-2148 5.10E+06 1.30E−01 24.5 TPP-2093 4.10E+06 9.00E−02 22.1 TPP-2149 8.40E+06 1.00E−01 12.1 TPP 2090 9.10E+06 1.10E−01 12.4 PDL-192(TPP-(1104) 1.00E+07 3.80E−02 3.7 136.1(TPP-2194) 3.84E+07 3.24E−02 0.8 18.3.3(TPP-2193) 1.64E+07 2.85E−02 1.7 P4A8(TPP-1324) 1.20E+06 2.70E−03 2.3 P3G5(TPP-2195) 2.31E+06 1.22E−03 0.5 P2D3(TPP-2196) 1.32E+06 5.64E−04 0.4 ITEM-1 3.80E+06 1.10E−02 2.9 TEM-4 2.80E+06 2.00E−03 0.7

It was determined that the antibodies of the invention bind TWEAKR with morate affinity (K_(D) 10-200 nM), whereas some comparative antibodies (e.g. PDL-192(TPP-1104), 136.1(TPP-2194), 18.3.3(TPP-2193), P4A8(TPP-1324), P3G5(TPP-2195), P2D3(TPP-2196), ITEM-1, ITEM-4) show high-affinity binding (0.7-3.7 nM). The sequences of the variable domains of the antibodies PDL-192, 136.1, 18.3.3, P4A8, P3G5 and P2D3 were obtained from the patent literature WO2009/020933 and WO2009/140177, and the sequences coding for the constant region of human IgG1 and murine IgG2 were added, resulting in full-length IgGs PDL-192(TPP-1104), 136.1(TPP-2194), 18.3.3(TPP-2193), P4A8(TPP-1324), P3G5(TPP-2195), P2D3(TPP-2196). The range of the affinities measured in this study agrees well with published data: for PDL-192, 18.3.3 and 136.1, KD values of 5.5, 0.2 and 0.7 nM have been published (WO2009/020933); for P4A8 2.6 nM (WO2009/140177). For comparison: the native ligand TWEAK binds TWEAKR with a K_(D) value of 0.8-2.4 nM (Immunity. 2001 November; 15(5):837-46; Biochem J. 2006 Jul. 15; 397(2):297-304; Arterioscler Thromb Vasc Biol. 2003 Apr. 1; 23(4):594-600).

As a result, it can be recorded that the antibodies of the invention (TPP-883, TPP-1538, TPP-2077, TPP-2084, TPP-2148, TPP-2093, TPP-2149 and TPP-2090) bind TWEAKR with morate affinity (K_(D) 10-200 nM).

Characterization of the Binding Epitope of TPP-2090 Using N- and C-Terminally Truncated Variants of the TWEAKR Ectodomain:

The alignment of the cysteine-rich domain of TWEAKR (amino acids 34-68) of different species (FIG. 1 ) shows that it is well conserved in all 6 species analysed. PDL-192 binds depending on R56 (WO2009/020933: FIG. 2B) and does therefore not bind to rat, pig and mouse TWEAKR. TPP-2090 binds depending on the conserved amino acid D47, and therefore binds to all species shown.

In a first approach to characterizing the binding epitope of the antibodies mentioned above, a N- and C-terminally truncated mutant of the TWEAKR ectodomain was generated and examined for its ability to bind the various anti-TWEAKR antibodies. Amino acids 28 to 33 were deleted N-terminally and amino acids 69 to 80 were deleted C-terminally, such that the cysteine-rich domains with disulphide bridges between Cys36-Cys49, Cys52-Cys67 and Cys55-Cys64 remained intact (compare FIG. 2 ). Both constructs, the full ectodomain 28-80 including N- and C-terminus and the truncated ectodomain 34-68, were expressed and purified as Fc fusion proteins TPP-2202 and TPP-2203, respectively.

To analyse the binding, 1 μg/ml of the corresponding dimeric TWEAKR Fc construct was coated, and 0.3 μg/ml and 0.08 μg/ml of biotinylated IgG were used as soluble binding partner. Detection was carried out using streptavidin-HRP and Amplex Red substrate. IgGs were biotinylated using an about 2-fold molar excess of biotin-LC-NHS (Pierce; Cat. No. 21347) according to the instructions of the manufacturer and desalted using Zeba desalting columns (Pierce; Cat. No. 89889). At all concentrations used of the soluble ligand, the antibodies of the present invention displayed saturated binding to both constructs, whereas the antibodies P4A8(TPP-1324), P3G5(TPP-2195) and Item-4 showed saturated binding only to the full-length ectodomain, but worsened binding to the N- and C-terminally truncated constructs (FIG. 3 and FIG. 4 ). This shows that the binding epitope of the antibodies of the present invention is located in the cysteine-rich domain between amino acids 34-68. To analyse whether the N-terminus or the C-terminus of the TWEAKR ectodomain is required for P4A8(TPP-1324) and P3G5(TPP-2195) binding, a monomeric ectodomain having the C-terminal deletion of amino acids 69 to 80 was generated. Binding of P4A8(TPP-1324) and P3G5(TPP-2195) to the C-terminally truncated TWEAKR ectodomain is likewise worsened, whereas the antibodies of the present invention show saturated binding (FIG. 5 ).

TABLE AK-9 List of recombinant antigens used in the ELISA analysis for epitope profiling Nomenclature Description SEQ ID NO TPP-2202 TWEAKR-ECD-28-80-hIgGFc-His 139 TPP-2203 TWEAKR-ECD-34-68-hIgGFc-His 140 TPP-1984 hTNFRSF12 amino acids 28-68-CT-His 141

TABLE AK-10 List of antibodies used in the ELISA analysis for epitope profiling SEQ ID NO Nomenclature Description Light chain Heavy chain P3G5(TPP-2195) murine IgG2a 121 122 P4A8(TPP-1324) human IgG1 125 126 136.1(TPP-2194) murine IgG2a 123 124 PDL-192(TPP-1104) human IgG1 127 128 TPP 2090 human IgG1 1 2 TPP-2084 human IgG1 41 42

Thus, the binding epitope of TPP-2090, TPP-2084, PDL-192(TPP-1104) and 136.1(TPP-2194) in the cysteine-rich domain and the binding epitope of P4A8(TPP-1324) and P3G5(TPP-2195) are located at least partially outside of the cysteine-rich domain.

Effect of TWEAKR-Fc Muteins on the Antibody Affinity

To examine the binding characteristics of the antibodies of the invention in more detail, certain muteins of TWEAKR suggested to be of relevance for the activity of known agonistic antibodies (WO2009/140177) were investigated. To this end, the full-length ectodomain (amino acids 28-80) having the individual amino acid substitutions below were expressed and purified as Fc fusion proteins: T33Q; S40R; W42A; M50A; R56P; H60K; L65Q.

TABLE AK-11 List of recombinant proteins used in the ELISA analysis for mutein binding Nomen- SEQ clature Description ID NO TPP-1990 hTNFRSF12 amino acids a28-80-L65Q-hIgG1-Fc 142 TPP-1989 hTNFRSF12 amino acids a28-80-H60K-hIgG1-Fc 143 TPP-2683 hTNFRSF12 amino acids a28-80-R56P-hIgG1-Fc 144 TPP-1988 hTNFRSF12 amino acids a28-80-M50A-hIgG1-Fc 145 TPP-1985 hTNFRSF12 amino acids a28-80-W42A-hIgG1-Fc 146 TPP-1987 hTNFRSF12 amino acids a28-80-S40R-hIgG1-Fc 147 TPP-1986 hTNFRSF12 amino acids a28-80-T33Q-hIgG1-Fc 148 TPP-599 hTNFRSF12 amino acids a28-80-hIgG1-Fc 138

To obtain dose-reaction data, the different TWEAKR-Fc muteins were coated at a low concentration (62 ng/ml) onto a 384-well Maxisorb ELISA plate, and a serial 2-fold dilution of biotinylated IgG beginning with a concentration of 100 nM was used as a soluble binding partner. Detection was carried out using streptavidin-HRP and Amplex Red. The IgGs examined were TPP-2090 and TPP-2084 of the present invention, PDL-192, 136.1 and 18.3.3 of WO2009/020933, P4A8 and P3G5 of WO2009/140177, and ITEM-1 and ITEM-4 of Nakayama et al [Biochem Biophys Res Com 306: 819-825].

TABLE AK-12 List of antibodies used in the ELISA analysis for mutein binding SEQ ID NO Nomenclature Description Light chain Heavy chain P3G5(TPP-2195) murine IgG2a 121 122 P4A8(TPP-1324) human IgG1 125 126 136.1(TPP-2194) murine IgG2a 123 124 PDL-192(TPP-1104) human IgG1 127 128 18.3.3(TPP-2193) murine IgG2a 129 130 TPP 2090 human IgG1 1 2 TPP-2084 human IgG1 41 42

TABLE AK-13 List of commercially available antibodies used in the ELISA for mutein binding Nomenclature Description Cat. No. (Abcam) ITEM-1 murine IgG1 ab21359 ITEM-4 murine IgG1 ab21127

IgGs were biotinylated using an about 2-fold molar excess of biotin-LC-NHS (Pierce; Cat. No. 21347) according to the instructions of the manufacturer and desalted using Zeba desalting columns (Pierce; Cat. No. 89889). The dose-reaction data were fitted and the IC50s were determined. To illustrate the results, a table was generated; “−” marks IC50s over 50 nM, “+” marks IC50s in the range from 1 to 150 pM.

TABLE AK-14 Effect of muteins on antibody binding T33Q S40R W42A M50A R56P H60K L65Q WT TPP-2084 + + − + + + + + TPP-2090 + + − + + + + + PDL-192 + + − + − + + + (TPP-1104) 136.1 + + − + − + + + (TPP-2194) 18.3.3 + + − + − + + + (TPP-2193) P4A8 + + − + + + + + (TPP-1324) P3G5 + + − + + + + + (TPP-2195) ITEM1 + + − + − + + + ITEM4 + + − + + − + +

As already published, ITEM-4 shows worsened binding to the H60K mutein [WO2009/140177: FIG. 23F] and PDL-192 to the R56P mutein [WO2009/020933: FIG. 22B]. In contrast to published data, ITEM-1 shows worsened binding to R56P, and all antibodies to W42A [WO2009/140177: FIG. 23E, FIG. 23F]. These differences can be explained by the methods chosen.

In contrast to ITEM-1, ITEM-4, PDL-192, 136.1 and 18.3.3, the antibodies of the present invention bind independently of all substitutions except for W42A.

Alanine Scan of the Cysteine-Rich Domain:

An alanine scan of the cysteine-rich domain (amino acids 34-68) was carried out in order to locate the binding site of the antibodies of the invention. FIG. 6A shows that N- and C-terminally truncated variants of the full-length ectodomain of TWEAKR do not worsen binding of the antibodies of the invention. Accordingly, the binding epitope is located in the cysteine-rich domain. The substitutions below were introduced into the TWEAKR(34-68) Fc construct: S37A, R38A, S40A, S41A, W42A, S43A, D45A, D47A, K48A, D51A, S54A, R56A, R58A, P59A, H60A, S61A, D62A, F63A and L65A.

TABLE AK-15 List of TWEAKR mutein constructs for the alanine scan of the cysteine-rich domain Nomenclature Description SEQ ID NO TPP-2203 TweakR-ECD-34-68-hIgGFc-His 140 TPP-2625 TweakR-ECD-34-68-hIgGFc-His-L65A 149 TPP-2624 TweakR-ECD-34-68-hIgGFc-His-F63A 150 TPP-2623 TweakR-ECD-34-68-hIgGFc-His-D62A 151 TPP-2622 TweakR-ECD-34-68-hIgGFc-His-S61A 152 TPP-2621 TweakR-ECD-34-68-hIgGFc-His-H60A 153 TPP-2620 TweakR-ECD-34-68-hIgGFc-His-P59A 154 TPP-2619 TweakR-ECD-34-68-hIgGFc-His-R58A 155 TPP-2618 TweakR-ECD-34-68-hIgGFc-His-R56A 156 TPP-2617 TweakR-ECD-34-68-hIgGFc-His-S54A 157 TPP-2616 TweakR-ECD-34-68-hIgGFc-His-D51A 158 TPP-2615 TweakR-ECD-34-68-hIgGFc-His-K48A 159 TPP-2614 TweakR-ECD-34-68-hIgGFc-His-D47A 160 TPP-2613 TweakR-ECD-34-68-hIgGFc-His-D45A 161 TPP-2612 TweakR-ECD-34-68-hIgGFc-His-S43A 162 TPP-2611 TweakR-ECD-34-68-hIgGFc-His-W42A 163 TPP-2610 TweakR-ECD-34-68-hIgGFc-His-S41A 164 TPP-2609 TweakR-ECD-34-68-hIgGFc-His-S40A 165 TPP-2608 TweakR-ECD-34-68-hIgGFc-His-R38A 166 TPP-2607 TweakR-ECD-34-68-hIgGFc-His-S37A 167

These TWEAKR(34-68) Fc muteins were expressed in HEK293 cells. To obtain dose-reaction data, IgGs were coated at a concentration of 1 μg/ml onto a 384-well Maxisorp ELISA plate, and a serial 2-fold dilution of the supernatant comprising the TWEAKR mutein was used as soluble binding partner. Detection was carried out using anti-HIS-HRP and Amplex Red. The IgGs examined were TPP-2090 of the present invention, PDL-192 of WO2009/020933 and P4A8 of WO2009/140177.

TABLE AK-16 List of antibodies used for the alanine scan of the cysteine-rich domain SEQ ID NO Nomenclature Description Light chain Heavy chain P4A8(TPP-1324) human IgG1 125 126 PDL-192(TPP-1104) human IgG1 127 128 TPP 2090 human IgG1 1 2

To assess the relevance of the TWEAKR mutein for binding to various IgGs, a correlation blot at a certain mutein concentration was prepared. By way of example, FIG. 6B shows the correlation blots for the 8-fold diluted supernatants of the TWEAKR expression broth, with PDL-192(TPP-1104) on the X axis and TPP-2090 on the Y axis. The blot shows that binding of TPP-2090 was worsened by substitution D47A, and binding of PDL-192(TPP-1104) was worsened by substitution R56A. Binding to P4A8(TPP-1324) was demonstrated for none of the constructs, which agrees with the results obtained above (FIG. 6B). Thus, the P4A8 epitope is localized at least partially outside of the cysteine-rich domain. The dependencies identified for certain TWEAKR amino acids for antibody interaction correlates with the agonistic activity determined for these antibodies. The native ligand TWEAK shows an effective activation of the TWEAKR and binds depending on leucine 46 in the cysteine-rich domain of TWEAKR (Pellegrini et al, FEBS 280:1818-1829). P4A8 displays a very low agonistic activity and interacts at least partially with domains outside of the cysteine-rich domain of TWEAKR. PDL-192 displays a morate agonistic activity and binds depending on R56 to the cysteine-rich domain, but opposite the TWEAK ligand site. TPP-2090 and TWEAK binding depends on D47 and L46, respectively, and they therefore bind to a similar binding site (FIG. 7 ).

It can be concluded the antibodies of the invention (e.g. TPP-2090) bind to TWEAKR in a manner depending on D47.

The dependencies identified for certain TWEAKR amino acids for antibody interaction correlates with the agonistic activity determined for these antibodies. The native ligand TWEAK shows an effective activation of the TWEAKR and binds depending on leucine 46 in the cysteine-rich domain of TWEAKR (Pellegrini et al, FEBS 280:1818-1829). P4A8 displays a very low agonistic activity and interacts at least partially with domains outside of the cysteine-rich domain of TWEAKR. PDL-192 displays a morate agonistic activity and binds depending on R56 to the cysteine-rich domain, but opposite the TWEAK ligand site. Antibodies of the present invention (Example TPP-2090) bind in a manner depending on D47, and TWEAK binds in a manner depending on L46, and binds to a similar, but distinct, binding site (FIG. 7 ). Accordingly, the antibodies of the present invention displaying strong agonistic activity bind to a novel epitope (D47-dependent) for antibodies associated with very high agonistic activity. It is interesting to note that Michaelson et al. (see page 369, left column in Michaelson J S et al, MAbs. 2011 July-August; 3(4):362-75) gave an explanation for the fact that all agonistic antibodies examined by them have weaker agonistic activity than the natural ligand TWEAK. They conclude that reduced efficacy could be a function of the dimeric binding interaction of the antibodies with TWEAKR, with TWEAK probably entering into a trimeric interaction. It is therefore a surprising result that an antibody of the invention, in spite of its dimeric interaction with TWEAKR, has an even higher agonistic activity. This surprising activity is linked to the specific binding properties of the antibodies of the invention, i.e. the specific binding to D47 of TWEAKR. 

The invention claimed is:
 1. A compound of one of the formulae below:

or a salt, solvate, salt of a solvate, or epimer thereof, wherein: X₁ is N, X₂ is N and X₃ is C; or X₁ is N, X₂ is C and X₃ is N; or X₁ is CH or CF, X₂ is C and X₃ is N; or X₁ is NH, X₂ is C and X₃ is C; or X₁ is CH, X₂ is N and X₃ is C; L₁ is an organic group not cleavable in vivo; SG is an in vivo cleavable 2-8 oligopeptide group; Ry is —H, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —C(═O)—NH₂, or —NH₂; R¹ is —H, -MOD, or —(CH₂)₀₋₃Z; wherein: -MOD is the group —(NR¹⁰)n-(G1)o-G2-G3; wherein: R¹⁰ is —H or C₁-C₃ alkyl; G1 is —NH—C(═O)—, —C(═O)NH— or

G2 is a linear and/or branched hydrocarbon group which has 1 to 10 carbon atoms and which may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NR^(y)—, —NR^(y)C(═O)—, —C(═O)—NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—, —C(═O)—NR^(y)NR^(y)—, —C(═O)—, —CR^(x)═N—O—, and where the hydrocarbon chain is optionally substituted with —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CNNH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid; wherein: R^(y) is —H, phenyl, C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid; R^(x) is —H, C₁-C₃-alkyl or phenyl; G3 is —H or —COOH; wherein MOD has at least one —COOH group; n is 0 or 1; and o is 0 or 1; Z is —H, halogen, —OY³, —SY³, —NHY³, —C(═O)—NY¹Y² or —C(═O)—OY³, Y¹ is —H, —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ or —CH(CH₂W)Z′ Y² is —H, —NH₂, —(CH₂CH₂O)₀₋₃—(CH₂)₀₋₃Z′ or —CH(CH₂W)Z′, Y³ is —H or —(CH₂)₀₋₃Z′, wherein: W is —H or —OH, Z′ is —H, —NH₂, —SO₃H, —COOH, —NH—C(═O)—CH₂—CH₂—CH(NH₂)COOH or —(C(═O)—NH—CHY⁴)₁₋₃COOH, and  Y⁴ is linear or branched C₁₋₆-alkyl which is optionally substituted by —NH—C(═O)—NH₂,  or Y⁴ is an aryl or benzyl, which are each optionally substituted by —NH₂.
 2. The compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; wherein: L₁ is an organic group not cleavable in vivo, of the formula: —(NH)_(n)-G-; wherein: G represents a bond or an optionally substituted linear or branched hydrocarbon chain having 1 to 100 carbon atoms, comprising arylene groups, or linear, branched, or cyclic alkylene groups, wherein the arylene or alkylene groups may be interrupted once or more than once by one or more of the groups —O—, —S—, —S(═O)—, —S(═O)₂—, —NH—, —C(═O)—, —NMe-, —NHNH—, —S(═O)₂—NHNH—, —NH—C(═O)—, —C(═O)—NH—, —C(═O)—NHNH— and a 5- to 10-membered aromatic or non-aromatic heterocycle having up to 4 heteroatoms selected from the group consisting of —N—, —O— and —S—, —S(═O)— or —S(═O)₂—; and n is 0 or
 1. 3. The compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; of one of the formulae below:

or a salt, solvate, salt of a solvate, or epimer thereof; wherein: X₁ is CH, X₂ is C, X₃ is N; L₁ is an organic group not cleavable in vivo; and Ry is —H, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —C(═O)—NH₂, or —NH₂.
 4. The compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; of the formula below:

or a salt, solvate, salt of a solvate, or epimer thereof; wherein: X₁ is CH, X₂ is C, X₃ is N; L₁ is an organic group not cleavable in vivo; SG is an in vivo cleavable 2-4 oligopeptide group, which can be cleaved by legumain or cathepsin; Ry is —H, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —C(═O)—NH₂, or —NH₂; and R¹ is —H, —COOH, or -MOD; wherein: MOD is —(NR¹⁰)_(n)-(G1)_(o)-G2-G3; R¹⁰ is —H or C₁-C₃-alkyl; and n is 0 or 1; G1 is —NH—C(═O)— or —C(═O)NH—; and o is 0 or 1; G2 is a straight-chain and/or branched hydrocarbon group which has 1 to 10 carbon atoms and which may be interrupted once or more than once identically or differently by the groups —O—, —S—, —S(═O)—, —S(═O)₂, —NR^(y)—, —NR^(y)C(═O)—, —C(═O)NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—, —C(═O)—NR^(y)NR^(y)—C(═O)—, —CR^(x)═N—O—, wherein the hydrocarbon chain including any side chains may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid; R^(x) is —H, C₁-C₃-alkyl or phenyl; and R^(y) is —H, phenyl, C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid; G3 is —H or —COOH.
 5. The compound of claim 4, or a salt, solvate, salt of a solvate, or epimer thereof; wherein: SG is an in vivo cleavable 2-4 oligopeptide group, which can be cleaved by legumain or cathepsin, having the formula: —CH(CH₂C(═O)—NH₂)—NH—P2-, —CH(CH₂C(═O)NH₂)—NH—P2-P3-, or —CH(CH₂C(═O)—NH₂)—NH—P2-(P3)₂-; wherein P2 is Ala, Gly, Val, Leu, Ile, Pro, Ser, Thr, citrulline or Asn; P3 is His, Pro, Ala, Val, Leu, Ile, Gly, Ser, citrulline or Gln, or the corresponding N-methyl-amino acid; Ry is —H, —C(═O)—NH-alkyl, —NH—C(═O)-alkyl, —C(═O)—NH₂, or —NH₂; R¹ is —H, —COOH, or -MOD; wherein: MOD is —(NR¹⁰)_(n)-(G1)_(o)-G2-G3; R¹⁰ is —H or C₁-C₃-alkyl; and n is 0 or 1; G1 is —NH—C(═O)— or —C(═O)NH—; and o is 0 or 1; G2 is a straight-chain and/or branched hydrocarbon group which has 1 to 10 carbon atoms and which may be interrupted once or more than once identically or differently by the groups —O—, —S—, —S(═O)—, —S(═O)₂, —NR^(y)—, —NR^(y)C(═O)—, —C(═O)NR^(y)—, —NR^(y)NR^(y)—, —S(═O)₂—NR^(y)NR^(y)—, —C(═O)—NR^(y)NR^(y)—C(═O)—, —CR′═N—O—, wherein the hydrocarbon chain including any side chains may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid; R^(x) is —H, C₁-C₃-alkyl or phenyl; and R^(y) is —H, phenyl, C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl, each of which may be substituted by —NH—C(═O)—NH₂, —COOH, —OH, —NH₂, NH—CN—NH₂, sulphonamide, sulphone, sulphoxide or sulphonic acid; G3 is —H or —COOH.
 6. The compound of claim 5, or a salt, solvate, salt of a solvate, or epimer thereof; wherein: R¹ is -MOD; MOD is -G1-G2-G3; G1 is —C(═O)—NH—; G2 is a linear hydrocarbon group which has 1 to 10 carbon atoms and is optionally interrupted by —C(═O)—NH— or —NHC(═O)—; and wherein the hydrocarbon chain is optionally substituted by —COOH or —NH₂; and G3 is —COOH.
 7. The compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; wherein L₁ contains 1-20 —CH₂CH₂O— groups.
 8. The compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; wherein L₁ is an organic group not cleavable in vivo selected from the group consisting of:

where r in each case independently of one another represents a number from 0 to
 20. 9. A compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; selected from the group consisting of:

or a salt, solvate, salt of a solvate, or epimer thereof, or a stereoisomer or mixture of stereoisomers thereof.
 10. The compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; wherein the salt is a trifluoroacetic acid salt.
 11. The compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; wherein the compound is configured to be recognized by a transglutaminase enzyme.
 12. A method of preparing an antibody-prodrug conjugate comprising reacting (i) a compound of claim 1, or a salt, solvate, salt of a solvate, or epimer thereof; (ii) an antibody comprising at least one glutamine residue which can be recognized by transglutaminase; and (iii) a transglutaminase protein; in a reaction medium, thereby producing the antibody-drug conjugate.
 13. The method of claim 12, wherein (i) is provided in 2- to 100-fold molar excess with respect to the antibody.
 14. The method of claim 12, wherein (ii) is selective for a cancer target molecule.
 15. The method of claim 12, wherein (iii) is a bacterial transglutaminase.
 16. The method of claim 12, wherein the reaction medium is a liquid solution.
 17. A compound, or a salt, solvate, salt of a solvate, or epimer thereof; selected from the group consisting of:

or a salt, solvate, salt of a solvate, or epimer thereof, or a stereoisomer or mixture of stereoisomers thereof.
 18. A method of preparing an antibody-prodrug conjugate comprising reacting (i) a compound of claim 17, or a salt, solvate, salt of a solvate, or epimer thereof; (ii) an antibody comprising at least one glutamine residue which can be recognized by transglutaminase; and (iii) a transglutaminase protein; in a reaction medium, thereby producing the antibody-drug conjugate. 